Increased lysine production by gene amplification using coryneform bacteria

ABSTRACT

The invention provides methods to increase the production of an amino acid from Corynebacterium species by way of the amplification of amino acid biosynthetic pathway genes in a host cell chromosome. In a preferred embodiment, the invention provides methods to increase the production of L-lysine in  Corynebacterium glutamicum  by way of the amplification of L-lysine biosynthetic pathway genes in a host cell chromosome. The invention also provides novel processes for the production of an amino acid by way of the amplification of amino acid biosynthetic pathway genes in a host cell chromosome and/or by increasing promoter strength. In a preferred embodiment, the invention provides processes to increase the production of L-lysine in  Corynebacterium glutamicum  by way of the amplification of L-lysine biosynthetic pathway genes in a host cell chromosome. The invention also provides novel isolated nucleic acid molecules for L-lysine biosynthetic pathway genes of  Corynebacterium glutamicum  such as a naturally occurring, feedback-sensitive form of aspartokinase (ask) resulting from a threonine to isoleucine mutation at amino acid residue  380  in the ask gene of ATCC 21529, aspartate-semialdehyde dehydrogenase (asd), dihydrodipicolinate synthase (dapA), dihydrodipicolinate reductase (dapB), diaminopimelate dehydrogenase (ddh), and diaminopimelate decarboxylase (lysA).

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims benefit to the filing dates of U.S.Provisional Application No. 60/184,130, filed Feb. 22, 2000; and U.S.Provisional Application No. 60/173,707, filed Dec. 30, 1999, each ofwhich is herein incorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to the areas of microbial genetics and recombinantDNA technology. The invention provides gene sequences, vectors,microorganisms, promoters and regulatory proteins useful for theproduction of L-lysine. The invention further provides a method toincrease the production of L-lysine.

2. Related Art

L-lysine is an important economic product obtained principally byindustrial-scale fermentation utilizing the Gram positiveCorynebacterium glutamicum, Brevibacterium flavum and Brevibacteriumlactofermentum (Kleemann, A., et. al., Amino Acids, in ULLMANN'sENCYCLOPEDIA OF INDUSTRIAL CHEMISTRY, vol. A2, pp.57-97, Weinham:VCH-Verlagsgesellschaft (1985)).

The stereospecificity of the amino acids produced by fermentation makesthe process advantageous compared with synthetic processes; generallyL-form amino acids are produced by the microbial fermentation process.The production of L-lysine and other amino acids through fermentation,utilizing cheap carbon sources such as molasses, glucose, acetic acidand ethanol, is a relatively inexpensive means of production.

Microorganisms employed in microbial processes for amino acid productionmay be divided into 4 classes: wild-type strain, auxotrophic mutant,regulatory mutant and auxotrophic regulatory mutant (K. Nakayama et al.,in NUTRITIONAL IMPROVEMENT OF FOOD AND FEED PROTEINS, M. Friedman, ed.,(1978), pp. 649-661).

Several fermentation processes utilizing various strains isolated forauxotrophic or resistance properties are known in the art for theproduction of L-lysine: U.S. Pat. No. 2,979,439 discloses mutantsrequiring amino acid supplementation (homoserine, or L-methionine andL-threonine); U.S. Pat. No. 3,700,557 discloses mutants having anutritional requirement for L-threonine, L-methionine, L-arginine,L-histidine, L-leucine, L-isoleucine, L-phenylalanine, L-cystine, orL-cysteine; U.S. Pat. No. 3,707,441 discloses a mutant having aresistance to an L-lysine analog; U.S. Pat. No. 3,687,810 discloses amutant having both an ability to produce L-lysine and a resistance tobacitracin, penicillin G or polymyxin; U.S. Pat. No. 3,708,395 disclosesmutants having a nutritional requirement for homoserine, L-threonine,L-threonine and L-methionine, L-leucine, L-isoleucine or mixturesthereof and a resistance to L-lysine, L-threonine, L-isoleucine oranalogs thereof, U.S. Pat. No. 3,825,472 discloses a mutant having aresistance to an L-lysine analog; U.S. Pat. No. 4,169,763 disclosesmutant strains of Corynebacterium that produce L-lysine and areresistant to at least one of aspartic analogs and sulfa drugs; U.S. Pat.No. 5,846,790 discloses a mutant strain able to produce L-glutamic acidand L-lysine in the absence of any biotin action-suppressing agent; andU.S. Pat. No. 5,650,304 discloses a strain belonging to the genusCorynebacterium or Brevibacterium for the production of L-lysine that isresistant to4-N-(D-alanyl)-2,4-diamino-2,4-dideoxy-L-arabinose2,4-dideoxy-L-arabinoseor a derivative thereof.

A considerable amount is known regarding the biochemical pathway forL-lysine synthesis in Corynebacterium species (recently reviewed by Sahmet al., Ann. N. Y. Acad. Sci. 782: 25-39 (1996)). Entry into theL-lysine pathway begins with L-aspartate (see FIG. 1), which itself isproduced by transamination of oxaloacetate. A special feature of C.glutamicum is its ability to convert the L-lysine intermediatepiperidine 2,6-dicarboxylate to diaminopimelate by two different routes,i.e. by reactions involving succinylated intermediates or by the singlereaction of diaminopimelate dehydrogenase. Overall, carbon flux into thepathway is regulated at two points: first, through feedback inhibitionof aspartate kinase by the levels of both L-threonine and L-lysine; andsecond through the control of the level of dihydrodipicolinate synthase.Therefore, increased production of L-lysine may be obtained inCorynebacterium species by deregulating and increasing the activity ofthese two enzymes.

More recent developments in the area of L-lysine fermentative productionin Corynebacterium species involve the use of molecular biologytechniques to augment L-lysine production. The following examples areprovided as being exemplary of the art: U.S. Pat. Nos. 4,560,654 and5,236,831 disclose an L-lysine producing mutant strain obtained bytransforming a host Corynebacterium or Brevibacterium speciesmicroorganism which is sensitive to S-(2-aminoethyl)-cysteine with arecombinant DNA molecule wherein a DNA fragment conferring bothresistance to S-(2-aminoethyl)-cysteine and L-lysine producing abilityis inserted into a vector DNA; U.S. Pat. No. 5,766,925 discloses amutant strain produced by integrating a gene coding for aspartokinase,originating from coryneform bacteria, with desensitized feedbackinhibition by L-lysine and L-threonine, into chromosomal DNA of aCorynebacterium species bacterium harboring leaky type homoserinedehydrogenase or a Corynebacterium species deficient in homoserinedehydrogenase gene; increased L-lysine production is obtained by geneamplification by way of a plasmid vector or utilizing a gene replacementstrategy. European Patent Applications EP 0 811 682 A2 and EP 0 854 189A2 both provide for increased production of L-lysine in Corynebacteriumspecies by way of gene amplification based on plasmid copy number.

SUMMARY OF THE INVENTION

It is an object of the invention to provide a method to increase theproduction of an amino acid in Corynebacterium species by amplifying,i.e., increasing, the number of a gene or genes of an amino acidbiosynthetic pathway in a host cell. Particularly preferredCorynebacterium species include Corynebacterium glutamicum,Brevibacterium flavum, and Brevibacterium lactofermentum.

It is an object of the invention to provide an isolated feed backresistant aspartokinase enzyme wherein the naturally occurring threonineamino acid residue 380 in the feedback sensitive form is changed toisoleucine in the ask gene of ATCC 21529. It is an object of theinvention to provide an isolated ask polypeptide comprising the aminoacid sequence of SEQ ID NO:2. It is another object of the invention toprovide an isolated polynucleotide molecule comprising a nucleotidesequence encoding the polypeptide sequence of SEQ ID NO:2. It is anotherobject of the invention to provide an isolated polynucleotide moleculecomprising a nucleic acid having the sequence of SEQ ID NO:1.

It is another object of the invention to provide a method comprisingtransforming a Corynebacterium species host cell with a polynucleotidemolecule comprising a nucleotide sequence encoding a polypeptidecomprising amino acid SEQ ID NO:2, wherein said isolated polynucleotidemolecule is integrated into said host cell's chromosome therebyincreasing the total number of said amino acid biosynthetic pathwaygenes in said host cell chromosome, and selecting a transformed hostcell. It is a further object of the invention to provide a methodcomprising screening for increased amino acid production. The method mayfurther comprise growing said transformed host cell in a medium andpurifying an amino acid produced by said transformed host cell.

In another embodiment, a method to increase the production of an aminoacid is a method comprising transforming a Corynebacterium species hostcell with an isolated nucleic acid molecule encoding the amino acidsequence of SEQ ID NO:2, wherein said isolated nucleic acid molecule isintegrated into said host cell's chromosome thereby increasing the totalnumber of said amino acid biosynthetic pathway genes in said host cellchromosome, and wherein said isolated nucleic acid molecule furthercomprises at least one of the following: a polynucleotide encoding aCorynebacterium species lysine pathway asd amino acid sequence; apolynucleotide encoding a Corynebacterium species lysine pathway dapAamino acid sequence; a polynucleotide encoding a Corynebacterium specieslysine pathway dapB amino acid sequence; a polynucleotide encoding aCorynebacterium species lysine pathway ddh amino acid sequence; apolynucleotide encoding a Corynebacterium species lysine pathway ′lysAamino acid sequence; a polynucleotide encoding a Corynebacterium specieslysine pathway lysA amino acid sequence; a polynucleotide encoding aCorynebacterium species lysine pathway ORF2 amino acid sequence, andselecting a transformed host cell. The method may further comprisegrowing said transformed host cell in a medium and purifying an aminoacid produced by said transformed host cell.

The term “′lysA ” refers to a truncated lysA gene or amino acid sequenceused by Applicants and described infra. The term “lysA” refers to thefull length lysA gene or amino acid sequence used by Applicants anddescribed infra.

It is another object of the invention to provide an isolatedpolynucleotide molecule comprising a nucleic acid molecule encoding theCorynebacterium glutamicum lysine pathway ask amino acid sequence of SEQID NO:2; and at least one additional Corynebacterium species lysinepathway gene selected from the group consisting of a nucleic acidmolecule encoding the asd polypeptide, a nucleic acid molecule encodingthe dapA polypeptide, a nucleic acid molecule encoding the dapBpolypeptide, a nucleic acid molecule encoding the ddh polypeptide, anucleic acid molecule encoding the ′lysA polypeptide, a nucleic acidmolecule encoding the lysA polypeptide and a nucleic acid moleculeencoding the ORF2 polypeptide. In a preferred embodiment of theinvention, the isolated polynucleotide molecule comprises pK184-KDABH′L.In another preferred embodiment of the invention, the isolated nucleicacid molecule comprises pK184-KDAB. In another preferred embodiment ofthe invention, the isolated nucleic acid molecule comprises pD2-KDABHL.In another preferred embodiment of the invention, the isolated nucleicacid molecule comprises pD11-KDABH′L.

It is another object of the invention to provide a host cell transformedwith an isolated polynucleotide molecule comprising a nucleotidesequence encoding an isolated polypeptide comprising the amino acidsequence of SEQ ID NO:2, wherein the isolated nucleic acid molecule isintegrated into the host cell's chromosome thereby increasing the totalnumber of amino acid biosynthetic pathway genes in the host cellchromosome. In one embodiment the polynucleotide further comprises atleast one additional Corynebacterium species lysine pathway geneselected from the group consisting of: a nucleic acid molecule encodingan asd polypeptide; a nucleic acid molecule encoding a dapA polypeptide;a nucleic acid molecule encoding a dapB polypeptide; a nucleic acidmolecule encoding a ddh polypeptide; a nucleic acid molecule encoding a′lysA polypeptide; a nucleic acid molecule encoding a lysA polypeptide;and a nucleic acid molecule encoding an ORF2 polypeptide.

In another embodiment, the polynucleotide further comprises a nucleicacid molecule encoding a polypeptide wherein said asd polypeptide is SEQID NO:4; said dapA polypeptide is SEQ ID NO:6; said dapB polypeptide isSEQ ID NO:8; said ddh polypeptide is SEQ ID NO:10; said ′lysApolypeptide is SEQ ID NO:21, said lysA polypeptide is SEQ ID NO:14; andsaid ORF2 polypeptide is SEQ ID NO:16.

In another embodiment, the polynucleotide further comprises a nucleicacid molecule wherein said asd polypeptide is SEQ ID NO:4; said dapApolypeptide is SEQ ID NO:6; said dapB polypeptide is SEQ ID NO:8, saidddh polypeptide is SEQ ID NO:10; said ′lysA polypeptide is SEQ ID NO:21;said lysA polypeptide is SEQ ID NO:14; and said ORF2 polypeptide is SEQID NO:16.

In another embodiment, the polynucleotide further comprises a nucleicacid molecule encoding the asd amino acid sequence of SEQ ID NO:4; anucleic acid molecule encoding the dapA amino acid sequence of SEQ IDNO:6; a nucleic acid molecule encoding the dapB amino acid sequence ofSEQ ID NO:8; and a nucleic acid molecule encoding the ORF2 amino acidsequence of SEQ ID NO:16.

In another embodiment, the polynucleotide further comprises a nucleicacid molecule encoding the asd amino acid sequence of SEQ ID NO:4; anucleic acid molecule encoding the dapA amino acid sequence of SEQ IDNO:6; a nucleic acid molecule encoding the dapB amino acid sequence ofSEQ ID NO:8; a nucleic acid molecule encoding the ddh amino acidsequence of SEQ ID NO:10; and a nucleic acid molecule encoding the ORF2amino acid sequence of SEQ ID NO:16.

In another embodiment, the polynucleotide further comprises a nucleicacid molecule encoding the asd amino acid sequence of SEQ ID NO:4; anucleic acid molecule encoding the dapA amino acid sequence of SEQ IDNO:6; a nucleic acid molecule encoding the dapB amino acid sequence ofSEQ ID NO:8; a nucleic acid molecule encoding the ddh amino acidsequence of SEQ ID NO:10; a nucleic acid molecule encoding the ′lysAamino acid sequence of SEQ ID NO:21; and a nucleic acid moleculeencoding the ORF2 amino acid sequence of SEQ ID NO:16.

In another embodiment, the polynucleotide further comprises a nucleicacid molecule encoding the asd amino acid sequence of SEQ ID NO:4; anucleic acid molecule encoding the dapA amino acid sequence of SEQ IDNO:6; a nucleic acid molecule encoding the dapB amino acid sequence ofSEQ ID NO:8; a nucleic acid molecule encoding the ddh amino acidsequence of SEQ ID NO:10; a nucleic acid molecule encoding the lysAamino acid sequence of SEQ ID NO:14; and a nucleic acid moleculeencoding the ORF2 amino acid sequence of SEQ ID NO:16.

In one embodiment, the transformed host cell is a Brevibacteriumselected from the group consisting of Brevibacterium flavum NRRL-B30218,Brevibacterium flavum NRRL-B30219, Brevibacterium lactofermentumNRRL-B30220, Brevibacterium lactofermentum NRRL-B30221, Brevibacteriumlactofermentum NRRL-B30222, Brevibacterium flavum NRRL-30234 andBrevibacterium lactofermentum NRRL-30235. In another embodiment, thehost cell is Escherichia coli DH5 α MCR NRRL-B30228. In anotherembodiment, the host cell is a C. glutamicum selected from the groupconsisting of C. glutamicum NRRL-B30236 and C. glutamicum NRRL-B30237.

It is another object of the invention to provide a method of producinglysine comprising culturing the host cells comprising the amino acidsequence of SEQ ID NO:2 wherein said host cells comprise one or more of(a) increased enzyme activity of one or more lysine biosynthetic pathwayenzymes compared to the genetically unaltered nonhuman host cell; (b)one or more copies of each gene encoding a lysine biosynthetic pathwayenzyme; and, (c) alteration of one or more transcription factorsregulating transcription of one or more genes encoding a lysinebiosynthetic pathway enzyme, wherein said host cell produces lysine insaid culture medium. In one embodiment of the invention, the increasedenzyme activity comprises overexpressing one or more genes encoding oneor more lysine biosynthetic pathway enzymes. In another embodiment ofthe invention the increased enzyme activity results from the activity ofone or more modified lysine biosynthetic pathway enzymes wherein saidenzyme modification results in a change in kinetic parameters,allosteric regulation, or both, compared to the enzyme lacking themodification. In another embodiment of the invention, alteration of oneor more transcription factors comprises one or more mutations intranscription inhibitor proteins, one or more mutations in transcriptionactivator proteins, or both, wherein said one or more mutationsincreases transcription of the target nucleotide sequence compared tothe transcription by said one or more transcription factors lacking saidalteration(s).

It is an object of the invention to provide an isolated polypeptide,wherein said polypeptide comprises an amino acid sequence having atleast 95% sequence identity to the amino acid sequence of SEQ ID NO:19.It is a further object of the invention to provide an isolatedpolypeptide comprising the amino acid sequence of SEQ ID NO:19. It is afurther object of the invention to provide an isolated polynucleotidecomprising a nucleic acid having the sequence of SEQ ID NO:18. It isanother object of the invention to provide host cell NRRL B30360.

It is an object of the invention to provide an isolated polypeptidewherein said polypeptide comprises a polypeptide having at least 95%sequence identity to the amino acid sequence of SEQ ID NO:21. It is afurther object of the invention to provide an isolated polypeptidecomprising the amino acid sequence of SEQ ID NO:21. It is a furtherobject of the invention to provide a polynucleotide molecule comprisinga nucleic acid having the sequence of SEQ ID NO:20.

It is an object of the invention to provide an isolated polynucleotidemolecule comprising a nucleotide sequence encoding the polypeptidecomprising the amino acid sequence of SEQ ID NO:2, further comprising apromoter sequence where said promoter sequence has at least 95% sequenceidentity to SEQ ID NO:17. It is a further object of the invention toprovide an isolated polynucleotide molecule comprising a nucleotidesequence encoding the polypeptide comprising the amino acid sequence ofSEQ ID NO:2, wherein the polynucleotide molecule further comprises thesequence of SEQ ID NO:17. It is a further object of the invention toprovide a host cell NRRL B30359.

Further objects and advantages of the present invention will be clearfrom the description that follows.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. A schematic of the L-lysine biosynthetic pathway inCorynebacterium glutamicum (Sahm et al., Ann. N. Y. Acad. Sci. 782:25-39 (1996)).

FIG. 2. The nucleotide sequence of ask (ATCC21529 sequence) (SEQ IDNO:1).

FIGS. 3A, B. The amino acid sequence of ask (ATCC 21529 sequence) (SEQID NOS:1-2).

FIG. 4. The nucleotide sequence of asd (ATCC 21529 sequence) (SEQ IDNO:3).

FIGS. 5A, B. The amino acid sequence of asd (ATCC 21529 sequence) (SEQID NOS:3-4).

FIG. 6. The nucleotide sequence of dapA (NRRL-B11474) (SEQ ID NO:5).

FIG. 7. The amino acid sequence of dapA (NRRL-B11474) (SEQ ID NOS:5-6).

FIG. 8. The nucleotide sequence of dapB (NRRL-B11474) (SEQ ID NO:7).

FIG. 9. The amino acid sequence of dapB (NRRL-B11474) (SEQ ID NOS:7-8).

FIG. 10. The nucleotide sequence of ddh (NRRL-B11474) (SEQ ID NO:9).

FIGS. 11A, B. The amino acid sequence of ddh (NRRL-B11474) (SEQ IDNOS:9-10).

FIG. 12. The nucleotide sequence of full length lysA (NRRL-B11474) (SEQID NO:11) used to obtain the truncated lysA (′lysA) nucleotide sequence.Underlined region annealed with lysA primer.

FIG. 13. The amino acid sequence of full length lysA (NRRL-B11474) (SEQID NO:12) comprising the truncated lysA (′lysA) amino acid sequence (SEQID NO:21). Underlined L: the last amino acid residue of lysA encoded inthe truncated PCR product.

FIG. 14. The nucleotide sequence of full length lysA (pRS6) (SEQ IDNO:13).

FIGS. 15A, B, C. The amino acid sequence of full length lysA (pRS6) (SEQID NOS:13-14).

FIG. 16. The nucleotide sequence of ORF2 (NRRL-B11474) (SEQ ID NO:15).

FIG. 17. The amino acid sequence of ORF2 (NRRL-B11474) (SEQ IDNOS:15-16).

FIG. 18. A schematic depiction of the construction of the pFC3-KDABHLand pFC3-KDABH′L lysine pathway gene constructs of the invention.

FIG. 19. Comparison of the aspartokinase (ask) amino acid sequence fromATCC13032, N13 and ATCC21529. A consensus sequence of the alignment isdepicted and alterations in the coding sequences are shown.

FIG. 20. The nucleotide sequence of the HpaI-PvuII fragment from pRS6(SEQ ID NO:17) comprising the P1 promoter.

FIGS. 21A, B. A schematic depiction of the construction of thepDElia2-KDABHP1L construct.

FIG. 22. A schematic depiction of the construction of thepDElia2_(FC5)-KDBHL construct.

FIG. 23. The nucleotide sequence of truncated ORF2 (SEQ ID NO:18).

FIG. 24. The amino acid sequence of truncated ORF2 (SEQ ID NOS:18-19).

FIG. 25. The nucleotide sequence of truncated LysA (′lysA)(NRRL-B11474)(SEQ ID NO:20).

FIG. 26. The amino acid sequence of truncated LysA (′LysA)(NRRL-B11474)(SEQ ID NO:21). Underlined L: the last amino acid residue of lysAencoded in the truncated product.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS A. Definitions

In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided. It is also to be noted that theterm “a” or “an” entity, refers to one or more of that entity, forexample, “a polynucleotide,” is understood to represent one or morepolynucleotides.

Allosteric Regulation. As used herein, the term refers to regulation ofenzyme activity through the binding of one or more ligands (allostericeffectors) to one or more binding sites. The ligands may be the samemolecule or different molecules. The molecules bind to sites on theenzyme other than the enzyme active site. As a result of the binding, aconformational change is induced in the enzyme which regulates affinityof the active site for its substrate or other ligands. Allostericeffectors may serve to enhance catalytic site substrate affinity(allosteric activators) or to reduce affinity (allosteric repressors).Allosteric effectors form the basis of metabolic control mechanisms suchas feedback loops, for example (See, Copeland, Robert A., in Enzymes. APractical Introduction to Structure, Mechanism, and Data Analysis, pages279-296, Wiley-VCH, New York (1996)).

Amino Acid Biosynthetic Pathway Genes. As used herein, the term “aminoacid biosynthetic pathway gene(s)” is meant to include those genes andgenes fragments encoding peptides, polypeptides, proteins, and enzymes,which are directly involved in the synthesis of amino acids. These genesmay be identical to those which naturally occur within a host cell andare involved in the synthesis of any amino acid, and particularlylysine, within that host cell. Alternatively, there may be modificationsor mutations of such genes, for example, the genes may containmodifications or mutations which do not significantly affect thebiological activity of the encoded protein. For example, the naturalgene may be modified by mutagenesis or by introducing or substitutingone or more nucleotides or by removing nonessential regions of the gene.Such modifications are readily performed by standard techniques.

Auxotroph. As used herein, the term refers to a strain of microorganismrequiring for growth an external source of a specific metabolite thatcannot be synthesized because of an acquired genetic defect.

Amino Acid Supplement. As used herein, the term refers to an amino acidrequired for growth and added to minimal media to support auxotrophgrowth.

Chromosomal Integration. As used herein, the term refers to theinsertion of an exogenous DNA fragment into the chromosome of a hostorganism; more particularly, the term is used to refer to homologousrecombination between an exogenous DNA fragment and the appropriateregion of the host cell chromosome.

Enhancers. As used herein, the term refers to a DNA sequence which canstimulate promoter activity and may be an endogenous element or aheterologous element inserted to enhance the level, i.e., strength of apromoter.

High Yield Derivative. As used herein, the term refers to strain ofmicroorganism that produces a higher yield from dextrose of a specificamino acid when compared with the parental strain from which it isderived.

Host Cell. As used herein, the term “host cell” is intended to beinterchangeable with the term “microorganism.” Where a difference isintended, the difference will be made clear.

Isolated Nucleic Acid Molecule. As used herein, the term is intended tomean a nucleic acid molecule, DNA or RNA, which has been removed fromits native environment. For example, recombinant DNA molecules containedin a vector are considered isolated for the purposes of the presentinvention. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe DNA molecules of the present invention. Isolated nucleic acidmolecules according to the present invention further include suchmolecules produced synthetically.

Lysine Biosynthetic Pathway Protein. As used herein, the term “lysinebiosynthetic pathway protein” is meant to include those peptides,polypeptides, proteins, and enzymes, which are directly involved in thesynthesis of lysine from aspartate. Also included are amino acidsequences as encoded by open reading frames (ORF), where the ORF isassociated with a lysine biosynthetic pathway operon. These proteins maybe identical to those which naturally occur within a host cell and areinvolved in the synthesis of lysine within that host cell.Alternatively, there may be modifications or mutations of such proteins,for example, the proteins may contain modifications or mutations whichdo not significantly affect the biological activity of the protein. Forexample, the natural protein may be modified by mutagenesis or byintroducing or substituting one or more amino acids, preferably byconservative amino acid substitution, or by removing nonessentialregions of the protein. Such modifications are readily performed bystandard techniques. Alternatively, lysine biosynthetic proteins may beheterologous to the particular host cell. Such proteins may be from anyorganism having genes encoding proteins having the same, or similar,biosynthetic roles.

Mutagenesis. As used herein, the term refers to a process whereby amutation is generated in DNA. With “random” mutagenesis, the exact siteof mutation is not predictable, occurring anywhere in the genome of themicroorganism, and the mutation is brought about as a result of physicaldamage caused by agents such as radiation or chemical treatment. rDNAmutagenesis is directed to a cloned DNA of interest, and it may berandom or site-directed.

Mutation. As used herein, the term refers to a one or more base pairchange, insertion or deletion, or a combination thereof, in thenucleotide sequence of interest.

Operably Linked. As used herein, the term “operably linked” refers to alinkage of polynucleotide elements in a functional relationship. Anucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For instance, apromoter or enhancer is operably linked to a coding sequence if itaffects the transcription of the coding sequence. Operably linked meansthat the DNA sequences being linked are typically contiguous and, wherenecessary, join two protein coding regions, contiguous and in readingframe. However, since enhancers generally function when separated fromthe promoter by several kilobases and intronic sequences may be ofvariable lengths, some polynucleotide elements may be operably linkedbut not contiguous.

Operon. As used herein, the term refers to a contiguous portion of atranscriptional complex in which two or more open reading framesencoding polypeptides are transcribed as a multi-cistronic messengerRNA, controlled by a cis-acting promoter and other cis-acting sequencesnecessary for efficient transcription, as well as additional cis actingsequences important for efficient transcription and translation (e.g.,mRNA stability controlling regions and transcription terminationregions). The term generally also refers to a unit of gene expressionand regulation, including the structural genes and regulatory elementsin DNA.

Parental Strain. As used herein, the term refers to a strain of hostcell subjected to some form of treatment to yield the host cell of theinvention.

Percent Yield From Dextrose. As used herein, the term refers to theyield of amino acid from dextrose defined by the formula [(g amino acidproduced/g dextrose consumed)*100]=% Yield.

Phenotype. As used herein, the term refers to observable physicalcharacteristics dependent upon the genetic constitution of a host cell.

Promoter. As used herein, the term “promoter” has its art-recognizedmeaning, denoting a portion of a gene containing DNA sequences thatprovide for the binding of RNA polymerase and initiation oftranscription and thus refers to a DNA sequence capable of controllingthe expression of a coding sequence or functional RNA. Promotersequences are commonly, but not always, found in the 5′ non-codingregions of genes. In general, a coding sequence is located 3′ to apromoter sequence. Sequence elements within promoters that function inthe initiation of transcription are often characterized by consensusnucleotide sequences. The promoter sequence consists of proximal andmore distal upstream elements (enhancers). As used herein, the term“endogenous promoter” refers to a promoter sequence which is a naturallyoccurring promoter sequence in that host microorganism. The term“heterologous promoter” refers to a promoter sequence which is anon-naturally occurring promoter sequence in that host microorganism.The heterologous occurring promoter sequence may be from any prokaryoticor eukaryotic organism. A synthetic promoter is a nucleotide sequence,having promoter activity, and not found naturally occurring in nature.

Promoters may be derived in their entirety from a native gene, or behybrid promoters. Hybrid promoters are composed of different elementsderived from different promoters found in nature, or even comprisesynthetic DNA segments. Hybrid promoters may be constitutive, inducibleor environmentally responsive.

Useful promoters include constitutive and inducible promoters. Many suchpromoter sequences are known in the art. See, for example, U.S. Pat.Nos. 4,980,285; 5,631,150; 5,707,928; 5,759,828; 5,888,783; 5,919,670,and, Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd Ed.,Cold Spring Harbor Press (1989). Other useful promoters includepromoters which are neither constitutive nor responsive to a specific(or known) inducer molecule. Such promoters may include those thatrespond to developmental cues (such as growth phase of the culture), orenvironmental cues (such as pH, osmoticum, heat, or cell density, forexample).

Examples of environmental conditions that may effect transcription byinducible promoters include anaerobic conditions, elevated temperature,or the presence of light. It is understood by those skilled in the artthat different promoters may direct the expression of a gene indifferent cell types, or in response to different environmentalconditions. Promoters which cause a gene to be expressed in most celltypes at most times are commonly referred to as “constitutivepromoters.” It is further recognized that since in most cases the exactboundaries of regulatory sequences have not been completely defined, DNAfragments of different lengths may have identical or similar promoteractivity.

Relative Growth. As used herein, the term refers to a measurementproviding an assessment of growth by directly comparing growth of aparental strain with that of a progeny strain over a defined time periodand with a defined medium.

Transcription factor. As used herein, the term “transcription factor”refers to RNA polymerases, and other proteins that interact with DNA ina sequence-specific manner and exert transcriptional regulatory effects.Transcriptional factors may be transcription inhibitory proteins ortranscription activator proteins. In the context of the presentinvention, binding sites for transcription factors (or transcriptioncomplexes) are often included in the transcriptional regulatoryelement(s).

Transcription factor recognition site. As used herein, a “transcriptionfactor recognition site” and a “transcription factor binding site” referto a polynucleotide sequence(s) or sequence motif(s) which areidentified as being sites for the sequence-specific interaction of oneor more transcription factors, frequently taking the form of directprotein-DNA binding. Typically, transcription factor binding sites canbe identified by DNA footprinting, gel mobility shift assays, and thelike, and/or can be predicted on the basis of known consensus sequencemotifs, or by other methods known to those of skill in the art.

Transcriptional Complex. As used herein, the term “transcriptional unit”or “transcriptional complex” refers to a polynucleotide sequence thatcomprises a structural gene (one or more exons), a cis-acting linkedpromoter and one or more other cis-acting sequences necessary forefficient transcription of the structural sequences, distal regulatoryelements necessary for appropriate transcription of the structuralsequences, and additional cis sequences important for efficienttranscription and translation (e.g., polyadenylation site, mRNAstability controlling sequences). See, for example U.S. Pat. No.6,057,299.

Transcriptional Regulatory Element. As used herein, the term“transcriptional regulatory element” refers to a DNA sequence whichactivates transcription alone or in combination with one or more otherDNA sequences. A transcriptional regulatory element can, for example,comprise a promoter, response element, negative regulatory element,silencer element, gene suppressor, and/or enhancer. See, for example,U.S. Pat. No. 6,057,299.

B. Microbiological and Recombinant DNA Methodologies

The invention as provided herein utilizes some methods and techniquesthat are known to those skilled in the arts of microbiology andrecombinant DNA technologies. Methods and techniques for the growth ofbacterial cells, the introduction of isolated DNA molecules into hostcells, and the isolation, cloning and sequencing of isolated nucleicacid molecules, etc., are a few examples of such methods and techniques.These methods and techniques are described in many standard laboratorymanuals, such as Davis et al., Basic Methods In Molecular Biology(1986), J. H. Miller, Experiments in Molecular Genetics, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (1972); J. H. Miller,A Short Course in Bacterial Genetics, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1992); M. Singer and P. Berg, Genes &Genomes, University Science Books, Mill Valley, Calif. (1991); J.Sambrook, E. F. Fritsch and T. Maniatis, Molecular Cloning: A LaboratoryManual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y. (1989); P. B. Kaufman et al., Handbook of Molecular and CellularMethods in Biology and Medicine, CRC Press, Boca Raton, Fla. (1995);Methods in Plant Molecular Biology and Biotechnology, B. R. Glick and J.E. Thompson, eds., CRC Press, Boca Raton, Fla. (1993); and P. F.Smith-Keary, Molecular Genetics of Escherichia coli, The Guilford Press,New York, N.Y. (1989), all of which are incorporated herein by referencein their entireties.

Unless otherwise indicated, all nucleotide sequences newly describedherein were determined using an automated DNA sequencer (such as theModel 373 from Applied Biosystems, Inc.). Therefore, as is known in theart, for any DNA sequence determined by this automated approach, anynucleotide sequence determined herein may contain some errors.Nucleotide sequences determined by automation are typically at leastabout 90% identical, more typically at least about 95% to at least about99.9% identical to the actual nucleotide sequence of the sequenced DNAmolecule. The actual sequence can be more precisely determined by otherapproaches including manual DNA sequencing methods well known in theart.

In certain embodiments, polynucleotides of the invention comprise anucleic acid, the sequence of which is at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% identical to a sequence selected from thegroup consisting of SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:20, or acomplementary sequence thereof.

By a polynucleotide comprising a nucleic acid, the sequence of which isat least, for example, 95% “identical” to a reference nucleotidesequence is intended that the nucleic acid sequence is identical to thereference sequence except that the nucleic acid sequence may include upto five mismatches per each 100 nucleotides of the reference nucleicacid sequence. In other words, to obtain a nucleic acid, the sequence ofwhich is at least 95% identical to a reference nucleic acid sequence, upto 5% of the nucleotides in the reference sequence may be deleted orsubstituted with another nucleotide, or a number of nucleotides up to 5%of the total nucleotides in the reference sequence may be inserted intothe reference sequence. The reference (query) sequence may be any one ofthe entire nucleotide sequences shown in SEQ ID NO:17, SEQ ID NO:18, orSEQ ID NO:20, or any fragment of any of these sequences, as describedinfra.

As a practical matter, whether any particular nucleic acid sequence isat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identicalto, for instance, a nucleotide sequence consisting of SEQ ID NO:17; SEQID NO.:18, or SEQ ID NO:20, or a complementary sequence thereof, can bedetermined conventionally using sequence analysis computer programs suchas a OMIGA® Version 2.0 for Windows, available from Oxford Molecular,Ltd. (Oxford, U.K.). OMIGA uses the CLUSTAL W alignment algorithm usingthe slow fall dynamic programming alignment method with defaultparameters of an open gap penalty of 10 and an extend gap penalty of5.0, to find the best alignment between two nucleotide sequences. Whenusing CLUSTAL W or any other sequence alignment program to determinewhether a particular sequence is, for instance, 95% identical to areference sequence according to the present invention, the parametersare set, of course, such that the percentage of identity is calculatedover the full length of the reference nucleotide sequence such thatgaps, mismatches, or insertions of up to 5% of the total number ofnucleotides in the reference sequence are allowed. Other sequenceanalysis programs, known in the art, can be used in the practice of theinvention.

This embodiment of the present invention is directed to polynucleotidescomprising a nucleic acid, the sequence of which is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a nucleic acidsequence of SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:20, or acomplementary sequence thereof, irrespective of whether they havefunctional activity. This is because even where a particularpolynucleotide does not have functional activity, one of skill in theart would still know how to use the nucleic acid molecule, for instance,as a hybridization probe, an S1 nuclease mapping probe, or a polymerasechain reaction (PCR) primer.

Preferred, however, are polynucleotides comprising a nucleic acid, thesequence of which is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% identical to a nucleic acid sequence of SEQ ID NO:17, SEQ IDNO:18 or SEQ ID NO:20, or a complementary sequence thereof, which do, infact, have functional activity in Corynebacterium species.

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a reference amino acid sequence of a polypeptide isintended that the amino acid sequence of the claimed polypeptide isidentical to the reference sequence except that the claimed polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the reference amino acid of the polypeptide. In otherwords, to obtain a polypeptide having an amino acid sequence at least95% identical to a reference amino acid sequence, up to 5% of the aminoacid residues in the reference sequence may be deleted or substitutedwith another amino acid, or a number of amino acids up to 5% of thetotal amino acid residues in the reference sequence may be inserted intothe reference sequence. These alterations of the reference sequence mayoccur at the amino or carboxy terminal positions of the reference aminoacid sequence or anywhere between those terminal positions, interspersedeither individually among residues in the reference sequence or in oneor more contiguous groups within the reference sequence.

As a practical matter, whether any particular polypeptide is at least80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to, forinstance, the amino acid sequence shown in SEQ ID NO:2 or to the aminoacid sequence encoded by a nucleic acid sequence can be determinedconventionally using known computer programs such the Bestfit program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, 575 Science Drive, Madison,Wis. 53711). When using Bestfit or any other sequence alignment programto determine whether a particular sequence is, for instance, 95%identical to a reference sequence according to the present invention,the parameters are set, of course, such that the percentage of identityis calculated over the full length of the reference amino acid sequenceand that gaps in homology of up to 5% of the total number of amino acidresidues in the reference sequence are allowed.

In a specific embodiment, the identity between a reference sequence(query sequence, a sequence of the present invention) and a subjectsequence, also referred to as a global sequence alignment, is determinedusing the FASTDB computer program based on the algorithm of Brutlag etal. (Comp. App. Biosci. 6:237-245 (1990)). Preferred parameters used ina FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2, MismatchPenalty=1, Joining Penalty=20, Randomization Group Length=0, CutoffScore=1, Window Size=sequence length, Gap Penalty=5, Gap SizePenalty=0.05, Window Size=500 or the length of the subject amino acidsequence, whichever is shorter. According to this embodiment, if thesubject sequence is shorter than the query sequence due to N- orC-terminal deletions, not because of internal deletions, a manualcorrection is made to the results to take into consideration the factthat the FASTDB program does not account for N- and C-terminaltruncations of the subject sequence when calculating global percentidentity. For subject sequences truncated at the N- and C-termini,relative to the query sequence, the percent identity is corrected bycalculating the number of residues of the query sequence that are N- andC-terminal of the subject sequence, which are not matched/aligned with acorresponding subject residue, as a percent of the total bases of thequery sequence. A determination of whether a residue is matched/alignedis determined by results of the FASTDB sequence alignment. Thispercentage is then subtracted from the percent identity, calculated bythe above FASTDB program using the specified parameters, to arrive at afinal percent identity score. This final percent identity score is whatis used for the purposes of this embodiment. Only residues to the N- andC-termini of the subject sequence, which are not matched/aligned withthe query sequence, are considered for the purposes of manuallyadjusting the percent identity score. That is, only query residuepositions outside the farthest N- and C-terminal residues of the subjectsequence. For example, a 90 amino acid residue subject sequence isaligned with a 100 residue query sequence to determine percent identity.The deletion occurs at the N-terminus of the subject sequence andtherefore, the FASTDB alignment does not show a matching/alignment ofthe first 10 residues at the N-terminus. The 10 unpaired residuesrepresent 10% of the sequence (number of residues at the N- andC-termini not matched/total number of residues in the query sequence) so10% is subtracted from the percent identity score calculated by theFASTDB program. If the remaining 90 residues were perfectly matched thefinal percent identity would be 90%. In another example, a 90 residuesubject sequence is compared with a 100 residue query sequence. Thistime the deletions are internal deletions so there are no residues atthe N- or C-termini of the subject sequence which are notmatched/aligned with the query. In this case the percent identitycalculated by FASTDB is not manually corrected. Once again, only residuepositions outside the N- and C-terminal ends of the subject sequence, asdisplayed in the FASTDB alignment, which are not matched/aligned withthe query sequence are manually corrected for. No other manualcorrections are made for the purposes of this embodiment.

C. Methods and Processes of the Invention

Various embodiments of the invention provide methods to increase theproduction of an amino acid and processes for the production of an aminoacid from a Corynebacterium species host cell. Particularly preferredCorynebacterium species of the methods and processes of the inventioninclude: Corynebacterium glutamicum, Brevibacterium flavum,Brevibacterium lactofermentum and other Corynebacteria and Brevibacteriaspecies known in the art.

As will be understood by those skilled in the art, the term“Corynebacterium species” includes those organisms previously identifiedin the literature as “Brevibacterium species,” for exampleBrevibacterium flavum and Brevibacterium lactofermentum which have nowbeen reclassified into the genus Corynebacterium (Int. J. Syst.Bacteriol. 41: 255 (1981)).

Amino acid biosynthetic pathway genes embodied by the methods andprocesses described herein include those for L-glycine, L-alanine,L-methionine, L-phenylalanine, L-tryptophan, L-proline, L-serine,L-threonine, L-cysteine, L-tyrosine, L-asparagine, L-glutamine,L-aspartic acid, L-glutamic acid, L-lysine, L-arginine, L-histidine,L-isoleucine, L-leucine, and L-valine biosynthesis. Particularlypreferred embodiments are drawn to biosynthetic pathway genes forL-lysine (Sahm et al., Ann. N. Y. Acad. Sci. 782: 25-39 (1996)),L-threonine, L-isoleucine, L-tryptophan, and L-valine.

By way of example, the amino acid pathway for L-lysine biosynthesis iswell known to skilled artisans of amino acid production inCorynebacterium species. Genes encoding the enzymes important for theconversion of L-aspartate to L-lysine include the ask, asd, dapA, dapB,ddh and lysA genes (FIG. 1). Thus, the invention provides herein forexemplary purposes only, specific embodiments utilizing L-lysinebiosynthetic pathway genes. Other embodiments drawn to the use ofbiosynthetic pathway genes for the synthesis of other amino acids arealso encompassed by the invention described herein.

The methods to increase the production of an amino acid and theprocesses for the production of an amino acid of the invention bothutilize a step requiring the transformation of an isolated nucleic acidmolecule into a Corynebacterium species host cell. As known to oneskilled in the art, transformation of an isolated nucleic acid moleculeinto a host cell may be effected by electroporation, transduction orother methods. These methods are described in the many standardlaboratory manuals referenced and incorporated herein.

The methods to increase the production of an amino acid and theprocesses for the production of an amino acid of the invention bothutilize a step requiring amplification of at least one amino acidbiosynthesis pathway gene. As known to one skilled in the art, the termamplification means increasing the number of a gene or genes of an aminoacid biosynthetic pathway by any means known in the art. Particularlypreferred means of amplification include: (1) the addition an isolatednucleic acid molecule comprising copies of a gene or genes of abiosynthetic pathway by insertion into the chromosome of a host cell,for example by homologous recombination, and (2) the addition anisolated nucleic acid molecule comprising copies of a gene or genes of abiosynthetic pathway into a host cell by way of a self-replicating,extra-chromosomal vector, for example, a plasmid.

Another method of the invention to increase the production of an aminoacid comprises increasing the expression of at least one amino acidbiosynthetic pathway gene. Preferred methods of increasing expressioncomprise using heterologous promoters, regulated promoters, unregulatedpromoters and combinations thereof.

Methods of inserting an isolated nucleic acid molecule into thechromosome of a host cell are known to those skilled in the art. Forexample, insertion of isolated nucleic acid molecules into thechromosome of Corynebacterium species may be done utilizing the pK184plasmid described by Jobling, M. et al., Nucleic Acids Research 18(17):5315-5316 (submitted 1990). Because these vectors lack a Corynebacteriumspecies origin of replication and contain a selectable marker such askanamycin (kan), cells will only be capable of growing under selectionif the vector has been inserted into the host cell chromosome byhomologous recombination.

In alternative embodiments, the invention also provides methods forincreasing amino acid production and processes for the production of anamino acid wherein biosynthetic pathway gene amplification isaccomplished through the introduction into a host cell of aself-replicating, extra-chromosomal vector, e.g., a plasmid, comprisingan isolated nucleic acid molecule encoding an amino acid biosyntheticpathway gene or genes. Suitable plasmids for these embodiments includepSR1 and other derivatives of pSR1 (Archer, J. et al., J. Gen.Microbiol. 139: 1753-1759 (1993)).

For various embodiments of the invention drawn to a method to increaseproduction of an amino acid, screening for increased production of anamino acid, for example L-lysine, may be determined by directlycomparing the amount of L-lysine produced in culture by aCorynebacterium species host strain to that of a Corynebacterium speciestransformed host strain in which an amino acid biosynthesis gene orgenes are amplified. The level of production of the amino acid of choicemay conveniently be determined by the following formula to calculate thepercent yield from dextrose: [(g amino acid/L/(g dextroseconsumed/L)]*100.

In one embodiment, the invention provides a method to increase theproduction of an amino acid comprising: (a) transforming aCorynebacterium species host cell with an isolated polynucleotidemolecule comprising a nucleotide sequence encoding a polypeptidecomprising the amino acid sequence of SEQ ID NO:2; (b) amplifying thenumber of at least one of the biosynthetic pathway genes for said aminoacid in the chromosome of said host cell; (c) selecting a transformedhost cell; and (d) screening for increased production of said amino acidfrom said transformed host cell relative to said host cell.

In a particularly preferred embodiment, the invention provides a methodto increase the production of an amino acid comprising transforming aCorynebacterium species host cell with an isolated polynucleotidemolecule comprising a nucleotide sequence encoding a polypeptidecomprising the amino acid sequence of SEQ ID NO:2; and furthercomprising at least one of the following: a nucleic acid moleculeencoding a Corynebacterium species lysine pathway asd amino acidsequence; a nucleic acid molecule encoding a Corynebacterium specieslysine pathway dapA amino acid sequence; a nucleic acid moleculeencoding a Corynebacterium species lysine pathway dapB amino acidsequence; a nucleic acid molecule encoding a Corynebacterium specieslysine pathway ddh amino acid sequence; a nucleic acid molecule encodinga Corynebacterium species lysine pathway ′lysA amino acid sequence; anucleic acid molecule encoding a Corynebacterium species lysine pathwaylysA amino acid sequence; and a nucleic acid molecule encoding aCorynebacterium species lysine pathway ORF2 amino acid sequence.

In another particular embodiment of the method, the isolatedpolynucleotide molecule further comprises at least one of the following:a nucleic acid molecule encoding the asd amino acid sequence of SEQ IDNO:4; a nucleic acid molecule encoding the dapA amino acid sequence ofSEQ ID NO:6; a nucleic acid molecule encoding the dapB amino acidsequence of SEQ ID NO:8; a nucleic acid molecule encoding the ddh aminoacid sequence of SEQ ID NO:10; a nucleic acid molecule encoding the′lysA amino acid sequence of SEQ ID NO:21; a nucleic acid moleculeencoding the lysA amino acid sequence of SEQ ID NO:14; and a nucleicacid molecule encoding the ORF2 amino acid sequence of SEQ ID NO:16.

In another particular embodiment of the method, the isolatedpolynucleotide molecule further comprises the following: a nucleic acidmolecule encoding the asd amino acid sequence of SEQ ID NO:4; a nucleicacid molecule encoding the dapA amino acid sequence of SEQ ID NO:6; anucleic acid molecule encoding the dapB amino acid sequence of SEQ IDNO:8; and a nucleic acid molecule encoding the ORF2 amino acid sequenceof SEQ ID NO:16.

In another particular embodiment of the method, the isolatedpolynucleotide molecule further comprises the following: a nucleic acidmolecule encoding the asd amino acid sequence of SEQ ID NO:4; a nucleicacid molecule encoding the dapA amino acid sequence of SEQ ID NO:6; anucleic acid molecule encoding the dapB amino acid sequence of SEQ IDNO:8; a nucleic acid molecule encoding the ddh amino acid sequence ofSEQ ID NO:10, and a nucleic acid molecule encoding the ORF2 amino acidsequence of SEQ ID NO:16.

In another particular embodiment of the method, the isolatedpolynucleotide molecule further comprises the following: a nucleic acidmolecule encoding the asd amino acid sequence of SEQ ID NO:4; a nucleicacid molecule encoding the dapA amino acid sequence of SEQ ID NO:6; anucleic acid molecule encoding the dapB amino acid sequence of SEQ IDNO:8; a nucleic acid molecule encoding the ddh amino acid sequence ofSEQ ID NO:10; a nucleic acid molecule encoding the ′lysA amino acidsequence of SEQ ID NO:21; and a nucleic acid molecule encoding the ORF2amino acid sequence of SEQ ID NO:16.

In another particular embodiment of the method, the polynucleotidemolecule further comprises the following: a nucleic acid moleculeencoding the asd amino acid sequence of SEQ ID NO:4; a nucleic acidmolecule encoding the dapA amino acid sequence of SEQ ID NO:6; a nucleicacid molecule encoding the dapB amino acid sequence of SEQ ID NO:8; anucleic acid molecule encoding the ddh amino acid sequence of SEQ IDNO:10; a nucleic acid molecule encoding the lysA amino acid sequence ofSEQ ID NO:14; and a nucleic acid molecule encoding the ORF2 amino acidsequence of SEQ ID NO:16.

In another embodiment of the method, the method further comprisesgrowing said transformed host cell in a medium; and purifying an aminoacid produced by said transformed host cell.

It is another object of the invention to provide an isolatedpolynucleotide molecule comprising the polynucleotide moleculecomprising a nucleotide sequence encoding the polypeptide comprising theamino acid sequence of SEQ ID NO:2; and at least one additionalCorynebacterium species lysine pathway gene selected from the groupconsisting of a nucleic acid molecule encoding an asd polypeptide; anucleic acid molecule encoding a dapA polypeptide; a nucleic acidmolecule encoding a dapB polypeptide; a nucleic acid molecule encoding addh polypeptide; a nucleic acid molecule encoding a ′lysA polypeptide; anucleic acid molecule encoding a lysA polypeptide; and a nucleic acidmolecule encoding an ORF2 polypeptide. In a preferred embodiment, saidasd polypeptide is SEQ ID NO:4; said dapA polypeptide is SEQ ID NO:6;said dapB polypeptide is SEQ ID NO:8; said ddh polypeptide is SEQ IDNO:10; said ′lysA polypeptide is SEQ ID NO:21; said lysA polypeptide isSEQ ID NO:14; and said ORF2 polypeptide is SEQ ID NO:16.

It is another object of the invention to provide an isolatedpolynucleotide molecule comprising the polynucleotide moleculecomprising a nucleotide sequence encoding the polypeptide comprising theamino acid sequence of SEQ ID NO 2; a nucleic acid molecule encoding theasd amino acid sequence of SEQ ID NO:4; a nucleic acid molecule encodingthe dapA amino acid sequence of SEQ ID NO:6; a nucleic acid moleculeencoding the dapB amino acid sequence of SEQ ID NO:8; and a nucleic acidmolecule encoding the ORF2 amino acid sequence of SEQ ID NO:16.

It is another object of the invention to provide an isolatedpolynucleotide molecule comprising the polynucleotide moleculecomprising a nucleotide sequence encoding the polypeptide comprising theamino acid sequence of SEQ ID NO:2; a nucleic acid molecule encoding theasd amino acid sequence of SEQ ID NO:4; a nucleic acid molecule encodingthe dapA amino acid sequence of SEQ ID NO:6; a nucleic acid moleculeencoding the dapB amino acid sequence of SEQ ID NO:8; a nucleic acidmolecule encoding the ddh amino acid sequence of SEQ ID NO:10; and anucleic acid molecule encoding the ORF2 amino acid sequence of SEQ IDNO:16.

It is another object of the invention to provide an isolatedpolynucleotide molecule comprising the polynucleotide moleculecomprising a nucleotide sequence encoding the polypeptide comprising theamino acid sequence of SEQ ID NO:2; a nucleic acid molecule encoding theasd amino acid sequence of SEQ ID NO:4; a nucleic acid molecule encodingthe dapA amino acid sequence of SEQ ID NO:6; a nucleic acid moleculeencoding the dapB amino acid sequence of SEQ ID NO:8, a nucleic acidmolecule encoding the ddh amino acid sequence of SEQ ID NO:10; a nucleicacid molecule encoding the ′lysA amino acid sequence of SEQ ID NO:21;and a nucleic acid molecule encoding the ORF2 amino acid sequence of SEQID NO:16.

It is another object of the invention to provide an isolatedpolynucleotide molecule comprising the polynucleotide moleculecomprising a nucleotide sequence encoding the polypeptide comprising theamino acid sequence of SEQ ID NO:2; a nucleic acid molecule encoding theasd amino acid sequence of SEQ ID NO:4; a nucleic acid molecule encodingthe dapA amino acid sequence of SEQ ID NO:6; a nucleic acid moleculeencoding the dapB amino acid sequence of SEQ ID NO:8; a nucleic acidmolecule encoding the ddh amino acid sequence of SEQ ID NO:10; a nucleicacid molecule encoding the lysA amino acid sequence of SEQ ID NO:14; anda nucleic acid molecule encoding the ORF2 amino acid sequence of SEQ IDNO:16.

It is a further object of the invention to provide an isolatedpolynucleotide molecule comprising pK184-KDAB. It is a further object ofthe invention to provide an isolated polynucleotide molecule comprisingpK184-KDABH′L. It is a further object of the invention to provide anisolated polynucleotide molecule comprising pD11-KDABH′L. It is afurther object of the invention to provide an isolated polynucleotidemolecule comprising pD2-KDABHL.

It is a further object of the invention to provide a vector comprisingthe isolated polynucleotide molecule comprising a nucleotide sequenceencoding a polypeptide comprising the amino acid sequence of SEQ ID NO2; and further comprising at least one additional Corynebacteriumspecies lysine pathway gene selected from the group consisting of anucleic acid molecule encoding an asd polypeptide; a nucleic acidmolecule encoding a dapA polypeptide; a nucleic acid molecule encoding adapB polypeptide; a nucleic acid molecule encoding a ddh polypeptide; anucleic acid molecule encoding a ′lysA polypeptide; a nucleic acidmolecule encoding a lysA polypeptide; and a nucleic acid moleculeencoding an ORF2 polypeptide.

It is a further object to provide a host cell comprising a vectorcomprising the isolated polynucleotide molecule comprising a nucleotidesequence encoding a polypeptide comprising the amino acid sequence ofSEQ ID NO 2; and further comprising at least one additionalCorynebacterium species lysine pathway gene selected from the groupconsisting of a nucleic acid molecule encoding an asd polypeptide; anucleic acid molecule encoding a dapA polypeptide; a nucleic acidmolecule encoding a dapB polypeptide; a nucleic acid molecule encoding addh polypeptide; a nucleic acid molecule encoding a ′lysA polypeptide; anucleic acid molecule encoding a lysA polypeptide; and a nucleic acidmolecule encoding an ORF2 polypeptide.

It is a further object to provide a host cell wherein said host cell isa Brevibacterium selected from the group consisting of Brevibacteriumflavum NRRL-B30218, Brevibacterium flavum NRRL-B30219, Brevibacteriumlactofermentum NRRL-B30220, Brevibacterium lactofermentum NRRL-B30221,Brevibacterium lactofermentum NRRL-B30222, Brevibacterium flavumNRRL-30234 and Brevibacterium lactofermentum NRRL-30235. In anotherembodiment, the host cell is Escherichia coli DH5 α MCR NRRL-B30228. Inanother embodiment, the host cell is a C. glutamicum selected from thegroup consisting of C. glutamicum NRRL-B30236 and C. glutamicumNRRL-B30237.

The invention provides processes for the production of an amino acid. Inone embodiment, the invention provides a process for producing an aminoacid comprising: (a) transforming a Corynebacterium species host cellwith an isolated nucleic acid molecule; (b) amplifying the number ofchromosomal copies of at least one of the biosynthetic pathway genes forsaid amino acid; (c) selecting a transformed host cell; (d) growing saidtransformed cell in a medium; and (e) purifying said amino acid.

The invention is also directed to an isolated polypeptide comprising theamino acid sequence of SEQ ID NO:19. In one embodiment of the invention,the polypeptide has at least 95% sequence identity to the amino acidsequence of SEQ ID NO:19. The invention is also directed to an isolatedpolynucleotide molecule comprising a nucleotide sequence encoding thepolypeptide of SEQ ID NO:19. In one embodiment, the isolatedpolynucleotide comprises a nucleic acid having the sequence of SEQ IDNO:18.

The invention is also directed to a vector comprising the polynucleotidemolecule comprising a nucleotide sequence encoding the polypeptidecomprising the amino acid sequence of SEQ ID NO:19. In one embodiment,the invention is directed to a host cell comprising a vector encoding apolypeptide comprising the amino acid sequence of SEQ ID NO:19. In oneembodiment, the host cell is NRRL B30360.

The invention is also directed to a method comprising transforming aCorynebacterium species host cell with the polynucleotide moleculecomprising a nucleotide sequence encoding a polypeptide comprising theamino acid sequence of SEQ ID NO:19, and selecting a transformed hostcell. In one embodiment, the method further comprises screening forincreased amino acid production. In a preferred embodiment, the aminoacid screened for is lysine. In one embodiment, the polynucleotidemolecule is integrated into said host cell's chromosome, therebyincreasing the total number of said amino acid biosynthetic pathwaygenes in said host cell chromosome.

In another embodiment, the polynucleotide molecule further comprises atleast one of the following: (a) a nucleic acid molecule encoding aCorynebacterium species lysine pathway ask amino acid sequence, (b) anucleic acid molecule encoding a Corynebacterium species lysine pathwayasd amino acid sequence; (c) a nucleic acid molecule encoding aCorynebacterium species lysine pathway dapA amino acid sequence; (d) anucleic acid molecule encoding a Corynebacterium species lysine pathwaydapB amino acid sequence; (e) a nucleic acid molecule encoding aCorynebacterium species lysine pathway ddh amino acid sequence; (f) anucleic acid molecule encoding a Corynebacterium species lysine pathway′lysA amino acid sequence; (g) a nucleic acid molecule encoding aCorynebacterium species lysine pathway lysA amino acid sequence; and,(h) a nucleic acid molecule encoding an ORF2 polypeptide having SEQ IDNO:16. In this embodiment, the method further comprises screening forincreased amino acid production. In another embodiment, the amino acidscreened for is lysine.

In another embodiment of the method, the polynucleotide molecule furthercomprises: (a) a nucleic acid molecule encoding the ask amino acidsequence having SEQ ID NO:2; (b) a nucleic acid molecule encoding aCorynebacterium species lysine pathway asd amino acid sequence; (c) anucleic acid molecule encoding a Corynebacterium species lysine pathwaydapB amino acid sequence; (d) a nucleic acid molecule encoding aCorynebacterium species lysine pathway ddh amino acid sequence; and, (e)a nucleic acid molecule encoding a Corynebacterium species lysinepathway lysA amino acid sequence. In one embodiment of this method, themethod further comprises screening for increased amino acid production.

The invention is also directed to an isolated polypeptide comprising theamino acid sequence of SEQ ID NO:21. In one embodiment, the polypeptidehas at least 95% sequence identity to the amino acid sequence of SEQ IDNO:21. The invention also comprises an isolated polynucleotide moleculecomprising a nucleotide sequence encoding the polypeptide comprising theamino acid sequence having at least 95% sequence identity to the aminoacid sequence of SEQ ID NO:21. The invention is further comprises apolynucleotide molecule comprising a nucleic acid having the sequence ofSEQ ID NO:20. In one embodiment the invention comprises a vectorcomprising the polynucleotide molecule comprising a nucleotide sequenceencoding the polypeptide comprising the amino acid sequence having atleast 95% sequence identity to the amino acid sequence of SEQ ID NO:21.The invention further comprises a host cell comprising the vectorcomprising the polynucleotide molecule comprising a nucleotide sequenceencoding the polypeptide comprising the amino acid sequence having atleast 95% sequence identity to the amino acid sequence of SEQ ID NO:21.

In one embodiment, the invention comprises a host cell selected from thegroup consisting of NRRL B30218, NRRL B30220 and NRRL B30222.

The invention is further directed to a method comprising transforming aCorynebacterium species host cell with a polynucleotide moleculecomprising a nucleotide sequence encoding the polypeptide comprising theamino acid sequence having at least 95% sequence identity to the aminoacid sequence of SEQ ID NO:21, and selecting a transformed host cell.The method further comprises screening for increased amino acidproduction; in particular, for lysine production. In one embodiment, thepolynucleotide molecule is integrated into said host cell's chromosome,thereby increasing the total number of said amino acid biosyntheticpathway genes in said host cell chromosome. In one embodiment the methodfurther comprises a polynucleotide molecule further comprising at leastone of the following: (a) a nucleic acid molecule encoding aCorynebacterium species lysine pathway ask amino acid sequence; (b) anucleic acid molecule encoding a Corynebacterium species lysine pathwayask amino acid sequence having SEQ ID NO. 2; (c) a nucleic acid moleculeencoding a Corynebacterium species lysine pathway asd amino acidsequence; (d) a nucleic acid molecule encoding a Corynebacterium specieslysine pathway dapA amino acid sequence; (e) a nucleic acid moleculeencoding a Corynebacterium species lysine pathway dapB amino acidsequence; (f) a nucleic acid molecule encoding a Corynebacterium specieslysine pathway ddh amino acid sequence; (g) a nucleic acid moleculeencoding a Corynebacterium species lysine pathway ORF2 amino acidsequence; and, (h) a nucleic acid molecule encoding a truncatedCorynebacterium species lysine pathway ORF2 amino acid sequence. In oneembodiment, the method further comprises screening for increased aminoacid production. In another embodiment, the amino acid screened for islysine.

Another embodiment of the invention is also directed to an isolatedpolynucleotide molecule comprising a nucleotide sequence encoding thepolypeptide comprising the amino acid sequence of SEQ ID NO:2, whereinthe polynucleotide molecule further comprises a promoter sequence havingSEQ ID NO:17. In one embodiment, the promoter sequence has at least 95%sequence identity to SEQ ID NO:17. In one embodiment, the promotersequence having at least 95% sequence identity to SEQ ID NO:17 isoperably directly linked to the LysA gene. In another embodiment of theinvention, there is a vector comprising the isolated polynucleotidemolecule comprising a nucleotide sequence encoding the polypeptidecomprising the amino acid sequence of SEQ ID NO:2, wherein thepolynucleotide molecule further comprises a promoter sequence whereinsaid promoter sequence has at least 95% sequence identity to SEQ IDNO:17. In another aspect of the invention, there is a host cellcomprising the vector comprising the isolated polynucleotide moleculecomprising a nucleotide sequence encoding the polypeptide comprising theamino acid sequence of SEQ ID NO:2, wherein the polynucleotide moleculefurther comprises a promoter sequence having at least 95% sequenceidentity to SEQ ID NO:17. In one embodiment, the host cell is NRRLB30359.

The invention is also directed to a method comprising transforming aCorynebacterium species host cell with the polynucleotide moleculecomprising a nucleotide sequence encoding the polypeptide comprising theamino acid sequence of SEQ ID NO:2, wherein the polynucleotide moleculefurther comprises a promoter sequence having at least 95% sequenceidentity to SEQ ID NO:17, and selecting a transformed host cell. In oneembodiment, the method further comprises screening for increased aminoacid production. In another embodiment, the amino acid screened for islysine. In another embodiment of the method, the polynucleotide moleculeis integrated into said host cell's chromosome, thereby increasing thetotal number of amino acid biosynthetic pathway genes in said host cellchromosome. In another embodiment of the method, the polynucleotidemolecule further comprises at least one of the following: (a) a nucleicacid molecule encoding a Corynebacterium species lysine pathway asdamino acid sequence; (b) a nucleic acid molecule encoding aCorynebacterium species lysine pathway dapA amino acid sequence; (c) anucleic acid molecule encoding a Corynebacterium species lysine pathwaydapB amino acid sequence; (d) a nucleic acid molecule encoding aCorynebacterium species lysine pathway ddh amino acid sequence; (e) anucleic acid molecule encoding a Corynebacterium species lysine pathwayORF2 amino acid sequence; (f) a nucleic acid molecule encoding atruncated Corynebacterium species lysine pathway ORF2 amino acidsequence; (g) a nucleic acid molecule encoding a Corynebacterium specieslysine pathway lysA amino acid sequence; and, (h) a nucleic acidmolecule encoding a truncated Corynebacterium species lysine pathwaylysA amino acid sequence. In this embodiment, the method furthercomprises screening for increased amino acid production; in particular,for lysine production.

In a different embodiment of the method, the polynucleotide moleculecomprises: (a) a nucleic acid molecule encoding a Corynebacteriumspecies lysine pathway asd amino acid sequence; (b) a nucleic acidmolecule encoding a Corynebacterium species lysine pathway dapA aminoacid sequence; (c) a nucleic acid molecule encoding a Corynebacteriumspecies lysine pathway dapB amino acid sequence; (d) a nucleic acidmolecule encoding a Corynebacterium species lysine pathway ddh aminoacid sequence; (e) a nucleic acid molecule encoding a Corynebacteriumspecies lysine pathway ORF2 amino acid sequence; and, (f a nucleic acidmolecule encoding a Corynebacterium species lysine pathway lysA aminoacid sequence. In this embodiment, the method further comprisesscreening for increased amino acid production. In a preferredembodiment, the amino acid is lysine.

A variety of media known to those skilled in the art may be used tosupport cell growth for the production of an amino acid. Illustrativeexamples of suitable carbon sources include, but are not limited to:carbohydrates, such as glucose, fructose, sucrose, starch hydrolysate,cellulose hydrolysate and molasses; organic acids, such as acetic acid,propionic acid, formic acid, malic acid, citric acid, and fumaric acid;and alcohols, such as glycerol. Illustrative examples of suitablenitrogen sources include, but are not limited to: ammonia, includingammonia gas and aqueous ammonia; ammonium salts of inorganic or organicacids, such as ammonium chloride, ammonium phosphate, ammonium sulfateand ammonium acetate; and other nitrogen-containing sources, includingmeat extract, peptone, corn steep liquor, casein hydrolysate, soybeancake hydrolysate, urea and yeast extract.

A variety of fermentation techniques are known in the art which may beemployed in processes of the invention drawn to the production of aminoacids. Generally, amino acids may be commercially produced from theinvention in fermentation processes such as the batch type or of thefed-batch type. In batch type fermentations, all nutrients are added atthe beginning of the fermentation. In fed-batch or extended fed-batchtype fermentations one or a number of nutrients are continuouslysupplied to the culture, right from the beginning of the fermentation orafter the culture has reached a certain age, or when the nutrient(s)which are fed were exhausted from the culture fluid. A variant of theextended batch of fed-batch type fermentation is the repeated fed-batchor fill-and-draw fermentation, where part of the contents of thefermenter is removed at some time, for instance when the fermenter isfull, while feeding of a nutrient is continued. In this way afermentation can be extended for a longer time.

Another type of fermentation, the continuous fermentation or chemostatculture, uses continuous feeding of a complete medium, while culturefluid is continuously or semi-continuously withdrawn in such a way thatthe volume of the broth in the fermenter remains approximately constant.A continuous fermentation can in principle be maintained for an infinitetime.

In a batch fermentation an organism grows until one of the essentialnutrients in the medium becomes exhausted, or until fermentationconditions become unfavorable (e.g., the pH decreases to a valueinhibitory for microbial growth). In fed-batch fermentations measuresare normally taken to maintain favorable growth conditions, e.g., byusing pH control, and exhaustion of one or more essential nutrients isprevented by feeding these nutrient(s) to the culture. The microorganismwill continue to grow, at a growth rate dictated by the rate of nutrientfeed. Generally a single nutrient, very often the carbon source, willbecome limiting for growth. The same principle applies for a continuousfermentation, usually one nutrient in the medium feed is limiting, allother nutrients are in excess. The limiting nutrient will be present inthe culture fluid at a very low concentration, often unmeasurably low.Different types of nutrient limitation can be employed. Carbon sourcelimitation is most often used. Other examples are limitation by thenitrogen source, limitation by oxygen, limitation by a specific nutrientsuch as a vitamin or an amino acid (in case the microorganism isauxotrophic for such a compound), limitation by sulphur and limitationby phosphorous.

The amino acid may be recovered by any method known in the art.Exemplary procedures are provided in the following: Van Walsem, H. J. &Thompson, M. C., J. Biotechnol. 59:127-132 (1997), and U.S. Pat. No.3,565,951, both of which are incorporated herein by reference.

The invention described herein provides isolated nucleic acid moleculescomprising at least one L-lysine amino acid biosynthesis gene. Unlessotherwise indicated, all nucleotide sequences described herein weredetermined using an automated DNA sequencer (such as the Model 373 fromApplied Biosystems, Inc.), and all amino acid, sequences of polypeptidesencoded by DNA molecules described herein were predicted by translationof the relative DNA sequence. Therefore, as is known in the art, for anyDNA sequence determined by this automated approach, any nucleotidesequence determined herein may contain some errors. Nucleotide sequencesdetermined by automation are typically at least about 90% identical,more typically at least about 95% to at least about 99.9% identical tothe actual nucleotide sequence of the sequenced DNA molecule. The actualsequence can be more precisely determined by other approaches includingmanual DNA sequencing methods well known in the art.

As is also known in the art, a single insertion or deletion in adetermined nucleotide sequence compared to the actual sequence willcause a frame shift in translation of the nucleotide sequence such thatthe predicted amino acid sequence encoded by a determined nucleotidesequence will be completely different from the amino acid sequenceactually encoded by the sequenced DNA molecule, beginning at the pointof such an insertion or deletion.

The invention provides several isolated nucleic acid molecules encodingcomprising at least one L-lysine amino acid biosynthesis pathway gene ofCorynebacterium glutamicum. More specifically, the invention providesthe following isolated nucleic acid molecules: the nucleotide sequenceof the ask gene from the strain ATCC 21529 (SEQ ID NO:1); the nucleotidesequence of the asd gene from the strain ATCC 21529 (SEQ ID NO:3); thenucleotide sequence of the dapA gene from the strain NRRL-B11474 (SEQ IDNO:5); the nucleotide sequence of the dapB gene from the strainNRRL-B11474 (SEQ ID NO:7); the nucleotide sequence of the ddh gene fromthe strain NRRL-B11474 (SEQ ID NO:9) and the nucleotide sequence of theORF2 gene from the strain NRRL-B11474 (SEQ ID NO:15). In addition, alsoprovided herein is the nucleotide sequence of lysA (SEQ ID NO:13) genefrom plasmid pRS6 (Marcel, T., et al., Molecular Microbiology 4:1819-1830 (1990)).

It is known in the art that amino acids are encoded at the nucleic acidlevel by one or more codons (code degeneracy). It is also known in theart that choice of codons may influence expression of a particular aminoacid sequence (protein, polypeptide, etc.). Thus, the invention isfurther directed to nucleic acid molecules encoding the ask amino acidsequence of SEQ ID NO:2 wherein the nucleic acid molecule comprises anycodon known to encode a particular amino acid. The invention is alsofurther directed to nucleic acid sequences (SEQ ID NOs: 1, 3, 5, 7, 9,11, 13, 15, 18 and 20) which comprise alternative codons in order tooptimize expression of the protein or polypeptide.

In addition to the above described isolated nucleic acid molecules, theinvention also provides isolated nucleic acid molecules comprising morethan one L-lysine Corynebacterium glutamicum biosynthesis gene. Suchisolated nucleic acid molecules are referred to as “cassette”constructs. These cassette constructs simplify for the practitioner thenumber of recombinant DNA manipulations required to achieve geneamplification of L-lysine biosynthesis genes.

In one embodiment drawn to a cassette construct, the invention providesan isolated nucleic acid molecule comprising: (a) a polynucleotideencoding the Corynebacterium glutamicum L-lysine pathway ask amino acidsequence of SEQ ID NO:2; and (b) at least one additional Corynebacteriumspecies L-lysine pathway gene selected from the group consisting of: (1)a polynucleotide encoding the asd polypeptide; (2) a polynucleotideencoding the dapA polypeptide; (3) a polynucleotide encoding the dapBpolypeptide; (4) a polynucleotide encoding the ddh polypeptide; (5) apolynucleotide encoding the ′lysA polypeptide, and (6) a polynucleotideencoding the ORF2 polypeptide.

The isolated nucleic acid molecules of the invention are preferablypropagated and maintained in an appropriate nucleic acid vector. Methodsfor the isolation and cloning of the isolated nucleic acid molecules ofthe invention are well known to those skilled in the art of recombinantDNA technology. Appropriate vectors and methods for use with prokaryoticand eukaryotic hosts are described by Sambrook et al., MolecularCloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y.,1989, the disclosure of which is hereby incorporated by reference.

A great variety of vectors can be used in the invention. Such vectorsinclude chromosomal, episomal and virus-derived vectors, e.g., vectorsderived from bacterial plasmids and from bacteriophage, as well asvectors derived from combinations thereof, such as those derived fromplasmid and bacteriophage genetic elements, such as cosmids andphagemids, all may be used in accordance with this aspect of the presentinvention. Generally, any vector suitable to maintain and propagate apolynucleotide in a bacterial host may be used in this regard.

A large numbers of suitable vectors and promoters for use in bacteriaare known, many of which are commercially available. Preferredprokaryotic vectors include plasmids such as those capable ofreplication in E. coli (such as, for example, pBR322, ColEl, pSC101,pACYC 184, πVX). Such plasmids are, for example, disclosed by Maniatis,T., et al., In: Molecular Cloning, A Laboratory Manual, Cold SpringHarbor Press, Cold Spring Harbor, N.Y. (1982)). The following vectorsare provided by way of example: pET (Novagen), pQE70, pQE60, pQE-9(Qiagen), pBs, phagescript, psiX174, pBlueScript SK, pBsKS, pNH8a,pNH16a, pNH18a, pNH46a (Stratagene), pTrc99A, pKK223-3, pKK233-3,pDR540, pRIT5 (Pharmacia).

Preferred vectors for the isolated nucleic acid molecules of theinvention include the pFC1 to pFC7 novel family of combinatorial cloningvectors (Lonsdale, D. M., et al., Plant Molecular Biology Reporter 13:343-345(1995)), the pK184 vector (Jobling, M. G. and Homes, R. K.,Nucleic Acid Research 18: 5315-5316 (1990)).

Another group of preferred vectors are those that are capable ofautonomous replication in Corynebacterium species. Such vectors are wellknown to those skilled in the art of amino acid production by way ofmicrobial fermentation, examples of which include pSR1, pMF1014α andvectors derived therefrom.

The invention provides an isolated amino acid sequence of the askpolypeptide of the strain ATCC 21529 (SEQ ID NO:2). The isolated askamino sequence disclosed herein possesses unique properties with respectto feedback resistance of ask enzyme activity to accumulated levels ofL-lysine and L-threonine in the culture medium. When compared to the DNAsequences of other Corynebacterium glutamicum ask-asd gene sequences,the invention discloses a threonine to isoleucine change at amino acidresidue 380 which results in resistance to feedback inhibition. Theinvention also includes other amino acid changes at residue 380 whichresult in decreased ask enzyme sensitivity to L-threonine and/orL-lysine.

In addition, and as described in more detail herein, the vector maycontain control regions that regulate as well as engender expression.Generally, such regions will operate by controlling transcription, suchas inducer or repressor binding sites and enhancers, among others.

Vectors of the present invention generally will include a selectablemarker. Such markers also may be suitable for amplification or thevectors may contain additional markers for this purpose. In this regard,vectors preferably contain one or more selectable marker genes toprovide a phenotypic trait for selection of transformed host cells. Suchmarkers include, but are not limited to, an antibiotic resistance genesuch as a chloramphenicol, ampicillin, or kanamycin resistance gene, oran autotrophic gene which allows the host cell to grow in the absence ofa nutrient for which the host cell strain is normally auxotrophic.

If the vector is intended to be maintained in the host cellextrachromosomally, it will contain, in addition and origin ofreplication which will allow it to replicate in the Corynebacteriumspecies host cell. Alternatively, if it is desired that the vectorintegrate into the Corynebacterium species chromosome, the vector isconstructed such that it cannot replicate in Corynebacterium. Forexample, such a vector might be capable of propagation in anotherorganism, for example, E. coli, but lack the proper origin ofreplication to be propagated in Corynebacterium. In another aspect ofthis embodiment, the vector is a shuttle vector which can replicate andbe maintained in more than one host cell species, for example, such ashuttle vector might be capable of replication in a Corynebacterium hostcell such as a C. glutamicum host cell, and also in an E. coli hostcell.

The invention further provides the following isolated the amino acidsequences: the amino acid sequence of the asd polypeptide of the strainATCC 21529 (SEQ ID NO:4); the amino acid sequence of the dapApolypeptide of the strain NRRL-B11474 (SEQ ID NO:6); the amino acidsequence of the dapB polypeptide of the strain NRRL-B11474 (SEQ IDNO:8); the amino acid sequence of the ddh polypeptide of the strainNRRL-B11474 (SEQ ID NO:10) and the amino acid sequence of the ORF2polypeptide of the strain NRRL-B11474 (SEQ ID NO:16). In addition, alsoprovided herein is the amino acid sequence of lysA (pRS6) (Marcel, T.,et al., Mol. Microbiol. 4: 819-830 (1990)) (SEQ ID NO:14).

In addition to the isolated polypeptide sequences defined by thespecific sequence disclosures disclosed above, the invention alsoprovides the amino acid sequences encoded by the deposited clones.

It will be recognized in the art that some amino acid sequences of theinvention can be varied without significant effect of the structure orfunction of the proteins disclosed herein. Variants included mayconstitute deletions, insertions, inversions, repeats, and typesubstitutions so long as enzyme activity is not significantly affected.Guidance concerning which amino acid changes are likely to bephenotypically silent can be found in Bowie, J. U., et al., “Decipheringthe Message in Protein Sequences: Tolerance to Amino AcidSubstitutions,” Science 247:1306-1310 (1990).

The strains of the invention may be prepared by any of the methods andtechniques known and available to those skilled in the art. Introductionof gene constructs of the invention into the host cell can be effectedby electroporation, transduction or other methods. These methods aredescribed in the many standard laboratory manuals referenced andincorporated herein.

Various embodiments of the invention provide strains with increasedL-lysine production as a result of gene amplification. By geneamplification is meant increasing the number of copies above the normalsingle copy number of an L-lysine biosynthesis pathway gene by a factorof 2, 3, 4, 5, 10, or more copies.

In one embodiment of the invention, the additional copies of theL-lysine biosynthesis pathway gene(s) may be integrated into thechromosome. Another embodiment of the invention provides that theadditional copies of the L-lysine biosynthesis pathway gene(s) arecarried extra-chromosomally. Amplifications by a factor of 5 or less maybe obtained by introducing the additional gene copies into thechromosome of the host strain by way of single event homologousrecombination. In a most preferred embodiment, the recombination eventresults in the introduction of one additional copy of the copy of thegene or genes of interest. If more than 5 copies of the genes aredesired, then the invention also provides for the use of multicopyplasmids carrying the recombinant DNA construct of the invention.

Representative examples of appropriate hosts for isolated nucleic acidmolecules of the invention include, but are not limited to, bacterialcells, such as C. glutamicum, Escherichia coli, Streptomyces andSalmonella typhimurium cells; and fungal cells, such as yeast cells.Appropriate culture media and conditions for the above-described hostcells are known in the art.

Particularly preferred host cells of the invention include:Corynebacterium glutamicum, Brevibacterium flavum and Brevibacteriumlactofermentum.

Applicants have deposited clones carrying the pK184-KDABH′L multi-geneconstructs at an acceptable International Depository Authority inaccordance with the Budapest Treaty on the International Recognition ofthe Deposit of Microorganisms for the Purposes of Patent Procedure. Thedeposits have been made with the Agricultural Research Service, CultureCollection (NRRL), 1815 North University Street, Peoria, Ill. 61604.Deposits made in which the pK184-KDAB or pK184-KDABH′L multi-geneconstructs have been integrated into the chromosome of a host cellinclude the following: (1) the pK184-KDAB plasmid, integrated into thechromosome, deposited as NRRL-B30219 and NRRL-B30221 on Sep. 16, 1999and (2) the pK184-KDABH′L plasmid, integrated into the chromosome,deposited as NRRL-B30218, NRRL-B30220, and NRRL-B30222 on Sep. 16, 1999.In addition, the pK184-KDABH′L multigene construct in a plasmidconfiguration, carried in E. coli DH5α MCR, was deposited as NRRL-B30228on Sep. 29, 1999, and the pK184-KDAB isolated plasmid in E. coli wasdeposited as NRRL-B30628 on Sep. 17, 2002. E. coli comprisingpD11-KDABH′L was deposited as NRRL-B30629 on Sep. 17, 2002. The six geneconstruct (pDElia2-KDABHL) was deposited in E. coli (NRRL-B30233) onDec. 16, 1999. C. glutamicum comprising pK184-KDABH′L was deposited asNRRL-B30236 on Dec. 16, 1999. C. glutamicum comprising pK184-KDABHL wasdeposited as NRRL-B30237 on Dec. 16, 1999. C. glutamicum comprisingpDELia2-KDABHP1L was deposited as NRRL-B30359 on Oct. 31, 2000.Brevibacterium flavum comprising pDElia2-KDABHL was deposited asNRRL-B30234 on Dec. 16, 1999. Brevibacterium lactofermentum comprisingpDElia2-KDABHL was deposited as NRRL-B30235 on Dec. 16, 1999.

It is an object of the invention to provide a method of producing lysinecomprising culturing the host cells comprising the amino acid sequenceof SEQ ID NO:2 wherein said host cells comprise one or more of: (a)increased enzyme activity of one or more lysine biosynthetic pathwayenzymes compared to the genetically unaltered host cell; (b) one or morecopies of each gene encoding a lysine biosynthetic pathway enzyme; and,(c) alteration of one or more transcription factors regulatingtranscription of one or more genes encoding a lysine biosyntheticpathway enzyme, wherein said host cell produces lysine in said culturemedium. In one embodiment of the method, said increased enzyme activitycomprises overexpressing one or more genes encoding one or more lysinebiosynthetic pathway enzymes. In one embodiment of the method, said oneor more genes are operably linked directly or indirectly to one or morepromoter sequences. In another embodiment of the method, said operablylinked promoter sequences are heterologous, endogenous, or hybrid. In apreferred embodiment of the method, said promoter sequences are one ormore of: a promoter sequence from the 5′ end of genes endogenous to C.glutamicum, a promoter sequence from plasmids that replicate in C.glutamicum, and, a promoter sequence from the genome of phage whichinfect C. glutamicum. In a preferred embodiment of the method, one ormore of said promoter sequences are modified. In another preferredembodiment, said modification comprises truncation at the 5′ end,truncation at the 3′ end, non-terminal insertion of one or morenucleotides, non-terminal deletion of one or more nucleotides, additionof one or more nucleotides at the 5′ end, addition of one or morenucleotides at the 3′ end, and, combinations thereof.

In another embodiment of the method, said increased enzyme activityresults from the activity of one or more modified lysine biosyntheticpathway enzymes wherein said enzyme modification results in a change inkinetic parameters, allosteric regulation, or both, compared to theenzyme lacking the modification. In one embodiment of the method, saidchange in kinetic parameters is a change in K_(m), V_(max) or both. Inanother embodiment of the method, said change in allosteric regulationis a change in one or more enzyme allosteric regulatory sites. In oneembodiment, said change in allosteric regulation is a change in theaffinity of one or more enzyme allosteric regulatory sites for theligand or ligands. The ligands may be the same or different. In oneembodiment, said enzyme modification is a result of a change in thenucleotide sequence encoding said enzyme. In one embodiment, said changein said nucleotide sequence is an addition, insertion, deletion,substitution, or a combination thereof, of one or more nucleotides.

In another embodiment of the method, said alteration of one or moretranscription factors comprises one or more mutations in transcriptioninhibitor proteins, one or more mutations in transcription activatorproteins, or both, wherein said one or more mutations increasestranscription of the target nucleotide sequence compared to thetranscription by said one or more transcription factors lacking saidalteration. In one embodiment, said one or more mutations is a change insaid nucleotide sequence encoding said transcription factor. In anotherembodiment, said change in said nucleotide sequence is an addition,insertion, deletion, substitution, or a combination thereof, of one ormore nucleotides.

All patents and publications referred to herein are expresslyincorporated by reference in their entirety.

EXAMPLES Example 1 Preparation of L-Lysine Pathway Multi-gene ConstructspK184-KDAB and pK184-KDABH′L

Applicants have created L-lysine amino acid biosynthetic pathwaymulti-gene constructs for the purpose of amplifying the number of one ormore of the genes of this pathway in the chromosome of Corynebacteriumspecies. Also, through careful study of the L-lysine biosynthesis genesof strain ATCC 21529, Applicants have identified an amino acid change ofthreonine to isoleucine at amino acid residue 380 of the ask gene ofATCC 21529. Compared to the DNA sequences of other Corynebacteriumglutamicum ask genes, a threonine to isoleucine change at amino acidresidue 380 was observed (FIG. 19), which is responsible for the unusualfeedback resistant properties with respect to aspartate kinase enzymeregulation.

The isolated nucleic acid molecules encoding L-lysine, amino acidbiosynthesis pathway genes utilized in the present invention are fromthe following sources:

Gene(s) Source ask-asd Strain ATCC 21529; dapA Strain NRRL B11474; dapBStrain NRRL B11474; ddh Strain NRRL B11474; lysA Plasmid pRS6 (Marcel,T., et al., Mol. Microbiol. 4: 819-830 (1990)) carrying the lysA geneisolated from strain AS019, which was derived from ATCC 13059; lysA NRRLB11474; lysA NRRL B11474 (full length); and, ORF2 Strain NRRL B11474.

As one skilled in the art would know, the invention is not limited tothe specific strain origins that Applicants present for the isolatednucleic acid molecules of the invention. Any strain of Corynebacteriumspecies, particularly that of Corynebacterium glutamicum, may beutilized for the isolation of nucleic acid molecules that will be usedto amplify the number of chromosomally located amino acid biosyntheticpathway genes. Particularly preferred strains include: NRRL-B11474, ATCC21799, ATCC 21529, ATCC 21543, and E12.

Methods and techniques common to the art of recombinant DNA technologywere used in making the multi-gene constructs of the invention, as maybe found in the many laboratory manuals cited and incorporated herein,for example as found in J. Sambrook, E. F. Fritsch and T. Maniatis,Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989).

The polymerase chain reaction (PCR) technique is used extensively in themaking of the multi-gene constructs of the invention. In a typicalreaction, the standard 10× stock solution (100 mM Tris-HCL, pH 8.3, 500mM KCL, 1.5 mM MgCl₂) is diluted to 1× for use. Typical reactionconditions were used for PCR amplication: 10 mM Tris, pH 8.3, 50 mM KCl,1.5 mM MgCl₂, 0.01% gelatin, 200 μM deoxynucleotides, 0.2-1.0 μM primersand 2.5 U/100 μl pfu polymerase. Standard cycling parameters were alsoemployed in PCR reactions: For 30 cycles, template denaturation wasperformed at 94° C. for 1 min; 55° C. annealing temperature wasperformed for 1 min (or annealing temperature appropriate for particularprimer pair); product extension was performed at 72° C. for 1 min (ifproduct is <500 bp), 3 min (if product is >500 bp); and at the end ofcycling, a final extension at 72° C. for 7 min was performed.

The primers utilized for cloning experiments included:

-   -   ask: 5′-GGGTACCTCGCGAAGTAGCACCTGTCAC-3′ (SEQ ID NO:22);    -   asd: 5′-GCGGATCCCCCATCGCCCCTCAAAGA-3′ (SEQ ID NO:23);    -   dapB: 5′-AACGGGCGGTGAAGGGCAACT-3′ (SEQ ID NO:24);    -   dapA: 5′-TGAAAGACAGGGGTATCCAGA-3′ (SEQ ID NO:25);    -   ddh: 5′-CCATGGTACCAAGTGCGTGGCGAG-3′ (SEQ ID NO:26);        -   5′-CCATGGTACCACACTGTTTCCTTGC-3′ (SEQ ID NO:27);    -   argS: 5′-CTGGTTCCGGCGAGTGGAGCCGACCATTCCGCGAGG-3′ (SEQ ID NO:28);        and    -   lysA: 5′-CTCGCTCCGGCGAGGTCGGAGGCAACTTCTGCGACG-3′ (SEQ ID NO:29),        a primer that anneals internally to lysA (about 500 bp upstream        to the end of lysA). ′LysA is a truncated form obtained from        lysA.

Applicants utilized standard PCR and subcloning procedures in cloningthe coding regions of ask-asd, dapB-ORF2-dapA, ddh, ′lysA, and lysA.Construction procedures and intermediate plasmids are described in FIG.18. Applicants performed the following steps (FIG. 18) in constructingthe following vectors used in the L-lysine biosynthetic pathway:

-   -   1. pGEMT-ask-asd: an approximately 2.6 Kb PCR product containing        the ask-asd operon of ATCC21529 using primers ask and asd was        cloned into pGEM-T (Promega pGEM-T vector systems);    -   2. pADM21: an approximately 1.3 Kb PCR product (with an        engineered KpnI site on both primers) of NRRL-B11474 ddh coding        region was cloned into pADM20;    -   3. pUC 18-ddh: an approximately 1.3 Kb KpnI fragment of pADM21        containing ddh (NRRL-B11474) was subcloned into pUC 18 at the        KpnI site;    -   4. pLIC 1.7-argS-′lysA: PCR product using template NRRL-B11474        genomic DNA and primers argS and lysA was cloned into pPMG-LIC        cloning vector (PharMingen);    -   5. pM4-dapB-ORF2-dapA.: an approximately 3 Kb PCR product using        primers dapB and dapA was cloned into pM4 at the XbaI site;    -   6. pFC3-ask-asd: an approximately 2.6 Kb NsiI-ApaI fragment of        pGEMT-ask-asd was cloned into pFC3 cut with PstI and ApaI;    -   7. pFC1-ddh: ˜1.3 Kb SalI-EcoRI fragment of pUC18-ddh was cloned        into pPC1 cut with SalI and EcoRI;    -   8. pFC1-ddh-′lysA: an approximately 1.5 Kb EcoRI fragment        (containing the truncated lysA DNA) of pLIC1.7-argS-′lysA was        cloned into pFC1-ddh at the EcoRI site;    -   9. pFC5-dapB-ORF2-dapA: an approximately 3.4 Kb BamHI-BglII        fragment of pM4-dapB-ORF2-dapA was cloned into pFC5 at the BamHI        site;    -   10. pFC5-dapB-ORF2-dapA-ddh-′lysA: ˜2.8 Kb NheI fragment of        pFC1-ddh-′lysA was cloned into pFC5-dapB-ORF2-dapA at the NheI        site;    -   11. pFC-3-ask-asd-dapB-ORF2-dapA-ddh-′lysA: ˜6.2 Kb NotI        fragment of pFC5-dapB-ORF2-dapA-ddh-′lysA was cloned into        pFC3-ask-asd at the NotI site;    -   12. pDElia9-ask-asd-dapB-ORF2-dapA-ddh-′lysA (pDElia9-KDABH′L):        ˜8.8 Kb PmeI fragment of pFC3-ask-asd-dapB-ORF2-dapA-ddh-′lysA        was cloned into pDElia9 at the EcoRV site; and    -   13. pK184-ask-asd-dapB-ORF2-dapA-ddh-′lysA (pK184-KDABH′L): an        approximately 8.8 Kb PmeI fragment of        pFC3-ask-asd-dapB-ORF2-dapA-ddh-′lysA was cloned into pK184 at        the HincII or SmaI site.    -   14. pFC5-ask-asd-dapB-ORF2-dapA (pFC5-KDAB): ˜2.6 Kb KpnI-SmaI        fragment of pFC3-ask-asd was cloned into pFC5-dapB-ORF2-dapA cut        with KpnI and SmaI.    -   15. pK184-ask-asd-dapB-ORF2-dapA (pK184-KDAB): ˜7 Kb KpnI-PmeI        fragment of pFC5-ask-asd-dapB-ORF2-dapA was cloned into pK184        cut with KpnI and HincII.

Thus, Applicants have made the following L-lysine multi-gene constructs:

-   -   1. pK184-KDABH′L, wherein “K” represents a nucleotide sequence        encoding the ask polypeptide; “D” represents a nucleotide        sequence encoding the asd polypeptide; “A” represents a        nucleotide sequence encoding the dapA polypeptide; “B”        represents a nucleotide sequence encoding the dapB polypeptide;        “H′” represents a nucleotide sequence encoding the ddh        polypeptide; and “′L” represents a nucleotide sequence encoding        part of the ′lysA polypeptide. This construct is referred to as        a truncated 6 gene construct. The pK184-KDABHL construct,        constructed infra, is referred to as a full length 6 gene        construct.    -   2. pK184-KDAB, wherein “K” represents a nucleotide sequence        encoding the ask polypeptide; “D” represents a nucleotide        sequence encoding the asd polypeptide; “A” represents a        nucleotide sequence encoding the dapA polypeptide; and “B”        represents a nucleotide sequence encoding the dapB polypeptide.        This construct is referred to as a 4 gene construct.

Both pK184-KDABH′L and pK184-KDAB, as do the other constructs discussedherein, comprise the nucleotide sequence encoding the ORF2 polypeptide.

It should be noted that in addition to the indicated polypeptidesequences encoded by the isolated nucleic acid sequences represented by“K”, “D”, “A”, “B,” “H,” “L” and “′L”, these isolated nucleic acidsequences also include native promoter elements for the operonsrepresented therein. Thus, the ask-asd sequences have been cloned in afashion that includes the respective native promoter elements, the dapAand dapB sequences, representing the operon dapB-ORF2-dapA, have beencloned in a fashion that includes the respective promoter elements; theddh sequence has been cloned in a fashion that includes the respectivenative promoter elements, and the lysA and ′lysA sequences have beencloned in a fashion that includes a native promoter element.

Alternative gene promoter elements may be utilized in the constructs ofthe invention. For example, known bacterial promoters suitable for thisuse in the present invention include the E. coli lacI and lacZpromoters, the T3 and T7 promoters, the gpt promoter, the lambda PR andPL promoters, the trp promoter, or promoters endogenous to the bacterialcells of the present invention. Other promoters useful in the inventioninclude regulated promoters, unregulated promoters and heterologouspromoters. Many such promoters are known to one of skill in the art. SeeSambrook, E. F. et al., Molecular Cloning: A Laboratory Manual, 2d ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).

Example 2 Two-Fold Amplification of L-lysine Amino Acid BiosynthesisPathway Genes

For exemplary purposes only, Applicants provide herein an examplewherein at least one L-lysine amino acid biosynthesis pathway gene isamplified by a factor of 2 by way of (a) the introduction of an isolatednucleic acid molecule into a Corynebacterium glutamicum host cell, and(b) the subsequent single crossover homologous recombination eventintroducing said isolated nucleic acid molecule into saidCorynebacterium glutamicum host cell chromosome.

As will be understood by those in the art, at least one or two or threeor four or five or six or seven or eight or nine or ten or more aminoacid biosynthesis pathway genes may be amplified, i.e., increased innumber, by a factor of at least one or two or three or four or five orsix or seven or eight or nine or ten fold with minor variations of theexample presented herein.

pK184-KDAB, pK184-KDABH′L and pD2-KDABHL (a full length 6 gene constructconstructed in Example 4) plasmids were used in the construction of highyield derivative cell lines of the invention. This was accomplished byway of introducing plasmid pK184-KDAB, pK184-KDABH′L and pD2-KDABHL DNAsinto a Corynebacterium species resulting in incorporation of pK184-KDAB,pK184-KDABH′L or pD2-KDABHL into the host cell chromosome via a singlecrossover homologous recombination event. Amplification of the aminoacid biosynthetic pathway genes by way of chromosomal integration of theplasmid constructs of the invention provided increased L-lysineproduction in several Corynebacterium species strains.

For cell transformation experiments with the isolated nucleic acidmolecules of the invention, the growth and preparation of competentcells may be done according to the following procedure: (1) picking afresh, single colony of Corynebacterium glutamicum and growing a cultureovernight in 10 mL CM (SM1) in a 250 mL shake flask at 30 degreesCelsius with agitation; (2) inoculating 200 mL of “Growth Media” withthe overnight culture to an optical density (O.D.) of 660 nm of 0.1 in a500 mL shake flask; (3) growing the culture at 30 degrees Celsius withagitation for 5-6 hours; (4) pouring the culture into a chilled, sealed,sterile 250 mL centrifuge bottle; Spin at 8-10K for ten minutes inRefrigerated Sorvall at 4 degrees Celsius; (5) pouring off thesupernatant thoroughly and resuspending the cell pellet in an equalvolume of ice-cold, sterile, deionized water; (6) centrifuging thesample again under the same conditions; (7) repeating the water washremembering to keep everything ice-cold; (8) pouring off the supernatantthoroughly and resuspending the cell pellet in 1 mL of ice-cold, sterile10% glycerol and transferring the cells to a chilled, sterile, 1.5 mLmicrocentrifuge tube; (9) spin the sample for 10 minutes in arefrigerated centrifuge; (10) pipetting off and discarding thesupernatant, and resuspending the pellet in two to three times thepellet volume (200-400 μL) of 10% glycerol; and (11) alliquoting, ifnecessary, the cells into chilled tubes and freezing at −70 Celsius.

pK184-KDAB, pK184-KDABH′L and pD2-KDABHL plasmid DNAs were introducedinto Corynebacterium glutamicum host cells by the followingelectroporation procedure: (1) pipetting 35 μL cell/glycerol solutiononto the side wall of a chilled 0.1 cm electrocuvette; (2) pipettingabout 2-4 μL of plasmid into the solution and mixing the sample bygentle pipetting up and down; (3) bringing the entire solution to thebottom of the electrocuvette by gentle tapping, avoiding the creation ofbubbles; (4) keeping the sample on ice until ready for the electroshockstep, wiping off any moisture on the outside of the electrocuvette priorto the electroshock administration, and shocking the cells one time at1.5 kV, 200Ω, 25 μF.

Cells are allowed to recover from electroporation by: (1) immediatelypipetting 1 mL of warm “Recovery Media” into the electrocuvette andthoroughly mixing the solution by pipetting; (2) incubating the solution(in the electrocuvette) at 30 degrees Celsius for at least three hoursfor antibiotic resistance expression and cell recovery and (3) platingon selection media and incubating at 30 degrees Celsius for 3 days.

Example 3 Screening and Selection of Strains with Improved L-LysineProduction

After 3 days of growth, single colonies of antibiotic resistant cellsare individually selected to determine if there is increased L-lysineproduction over that which is produced by the parental host cell strain.

Recipes for all media used in these experiments are found in Tables 1and 2. L-lysine production is determined on cultures of transformed,antibiotic resistant cells grown in shaker flasks. Briefly, seed media(Table 1), was dispensed in 20 ml aliquots into deep baffled 250 mlBellco shake flasks and autoclaved for 20 minutes. After cooling to roomtemperature, these seed flasks were then inoculated with the strain tobe tested and placed on a rotary shaker. They were incubated at 30degrees Celsius, shaking, overnight. The following morning, the opticaldensity (wavelength=660 nm) of each seed was recorded, and 2 ml of theculture from each seed flask was transferred to a 21 ml aliquot of FM3media, also in a deep baffled shake flask. These “main” flasks were thenreturned to the shaker and incubated at 30 degrees Celsius.

After 48 hours of incubation, 1 ml of main culture was removed from eachflask, and the flasks were promptly returned to the shaker. From the 1ml sample, optical density was determined by diluting 1:50 in 0.1N HClto dissolve the calcium carbonate present in the media. The remainder ofeach sample was then centrifuged to pellet cells and calcium carbonate.A 1:50 dilution of the supernatant was made in water and from thisdilution the dextrose concentration was determined. ExtracellularL-lysine concentrations were also determined at this time by HPLC.

High yield derivative cells may be conveniently identified bydetermining the percent yield from dextrose, i.e., the yield of aminoacid from dextrose defined by the formula [(g amino acid produced/gdextrose consumed)*100]=% yield. Results are presented below in whichthe parental strains E12, NRRL-B11474 and ATCC 21799 are transformedwith the L-lysine multi-gene isolated nucleic acid molecules of theinvention identified as pK184-KDA, pK184-KDABH′L and pD(Elia)2-KDABHL.The pD2-KDABHL construct was made as in Example 4.

lysine L-lysine titer yield Strain Tested (g/L) (%) Cell DepositNRRL-B11474 31 44 NRRL-B11474::pK184-KDAB 32 45.7 NRRL-B-30219NRRL-B11474::pK184-KDABH'L 36 51.8 NRRL-B-30218NRRL-B11474::pDElia2-KDABHL 38 54.6 NRRL-B-30234 E12 1.4 0.9E12::pK184-KDABH'L 26.8 38 NRRL-B-30236 E12::pDElia2-KDABHL 29.8 42.5NRRL-B-30237 ATC21799 26.8 36.9 ATC21799::pK184-KDAB 28.5 39NRRL-B-30221 ATC21799::pK184-KDABH'L 31 43 NRRL-B-30220ATC21799::pDElia2-KDABHL 36 50 NRRL-B-30235

Once high yield derivative cell lines are identified, the cell lines arefurther screened to determine that amplification of the amino acidbiosynthetic pathway genes has occurred. Amplification screening may beconveniently accomplished either by (1) standard southern blotmethodology to determine gene copy number or (2) by a determination ofthe total enzyme activity for enzymes encoded by the respectivebiosynthetic pathway genes of the isolated nucleic acid moleculeintroduced into the host cell.

A determination of gene copy number by Southern blot methodology may bedone utilizing standard procedures known in the art of recombinant DNAtechnology, as described in the laboratory manuals referenced andincorporated herein, for example as found in J. Sambrook, E. F. Fritschand T. Maniatis, Molecular Cloning: A Laboratory Manual, 2d ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).

TABLE 1 Seed Media, SM1 Ingredient Concentration (g/L) Sucrose 50Potassium Phosphate, Monobasic 0.5 Potassium Phosphate, Dibasic 1.5 Urea3.0 Magnesium Sulfate  5.0 × 10⁻¹ Polypeptone 20 Beef Extract 5.0 Biotin7.56 × 10⁻⁴ Thiamine  3.0 × 10⁻³ Niacinamide 1.25 × 10⁻¹ L-Methionine 5.0 × 10⁻¹ L-Threonine  2.5 × 10⁻¹ L-Alanine  5.0 × 10⁻¹ pH 7.3

TABLE 2 Main Media, FM3 Ingredient Concentration (g/L) Dextrose* 60Ammonium Sulfate 50 Potassium Phosphate, Monobasic   1.0 MagnesiumSulfate 4.0 × 10⁻¹ Manganese Sulfate 1.0 × 10⁻² Ferrous Sulfate 1.0 ×10⁻² Biotin 3.0 × 10⁻⁴ Calcium Carbonate 50 Corn Steep Liquor (dissolvedsolids) 20 pH (adjusted with KOH)   7.4 *Dextrose was added afterautoclaving

Example 4 Preparation of L-Lysine Pathway Multi-Gene Constructs

The invention further comprises additional L-lysine multi-geneconstructs constructed using the PCR technique. Standard PCR andsubcloning procedures were utilized, as described above, to generate5-gene constructs similar to those in Example 1. The constructs of thisexample comprise the antibiotic resistance gene, chloramphenicol acyltransferase (CAT). The CAT gene was operably linked to a Corynebacteriaphosphofructokinase promoter for expression in Corynebacteria.

The following steps were performed in constructing the followingconstructs containing the CAT gene:

-   -   1. pGEMT-ask-asd: ˜2.6 Kb PCR product containing the ask-asd        operon of ATCC21529 using primers ask and asd was cloned into        pGEM-T (Promega pGEM-T vector systems);    -   2. pUC18-ddh: ˜1.3 Kb KpnI fragment of pADM21 containing ddh        (NRRL B11474) was subcloned into pUC18 at the KpnI site;    -   3. pLIC1.7-argS-′lysA: ˜3 Kb PCR product using template BF100        genomic DNA and primers argS and lysA was cloned into pPMG-LIC        cloning vector (PharMingen);    -   4. pM4-dapB-ORF2-dapA: ˜3 Kb PCR product using primers dapB and        dapA was cloned into pM4 at the blunted XbaI site;    -   5. pFC3-ask-asd: ˜2.6 Kb NsiI-ApaI fragment of pGEMT-ask-asd was        cloned into pFC3 cut with PstI and ApaI;    -   6. pFC1-ddh: ˜1.3 Kb SalI-EcoRI fragment of pUC18-ddh was cloned        into pFC1 cut with SalI and EcoRI;    -   7. pFC1-ddh-′lysA: ˜1.5 Kb EcoRI fragment (containing the        truncated lysA DNA) of pLIC1.7-argS-′lysA was cloned into        pFC1-ddh at the EcoRI site;    -   8. pFC1-ddh-lysA: ˜2.1 Kb EcoRI-PstI fragment (containing the        intact lysA DNA) of pRS6 was cloned into pFC1-ddh cut with EcoRI        and PstI;    -   9. pFC5-dapB-ORF2-dapA: ˜3.4 Kb BamHI-BglII fragment of        pM4-dapB-ORF2-dapA was cloned into pFC5 at the BamHI site;    -   10. pFC5-dapB-ORF2-dapA-ddh-′lysA: ˜2.8 Kb NheI fragment of        pFC1-ddh-′lysA was cloned into pFC5-dapB-ORF2-dapA at the NheI        site;    -   11. pFC5-dapB-ORF2-dapA-ddh-lysA: ˜3.4 Kb NheI fragment of        pFC1-ddh-lysA was cloned into pFC5-dapB-ORF2-dapA at the NheI        site;    -   12. pFC3-ask-asd-dapB-ORF2-dapA-ddh-′lysA (pFC3-KDABH′L): ˜6.2        Kb NotI fragment of pFC5-dapB-ORF2-dapA-ddh-′lysA was cloned        into pFC3-ask-asd at the NotI site;    -   13. pFC3-ask-asd-dapB-ORF2-dapA-ddh-lysA (pFC3-KDABHL): ˜6.8 Kb        NotI fragment of pFC5-dapB-ORF2-dapA-ddh-lysA was cloned into        pFC3-ask-asd at the NotI site;    -   14. pK184-ask-asd-dapB-OPF2-dapA-ddh-′lysA (pk184-KDABH′L): ˜8.8        Kb PmeI fragment of pFC3-ask-asd-dapB-ORF2-dapA-ddh-′lysA was        cloned into pK184 at the HincII or SmaI site;    -   15. pDElia2-ask-asd-dapB-ORF2-dapA-ddh-lysA (pD2-KDABHL): ˜9.4        Kb PmeI fragment of pFC3-ask-asd-dapB-ORF2-dapA-ddh-lysA was        cloned into pDElia2 at the HincII site (contains the kan gene;        is a full length 6 gene construct);    -   16. pDElia11-ask-asd-dapB-ORF2-dapA-ddh-′lysA (pD11-KDABH′L):        ˜8.8 Kb PmeI fragment of pFC3-ask-asd-dapB-ORF2-dapA-ddh-′lysA        was cloned into pDElia11 at the HincII or SmaI site (contains        the CAT gene; is a truncated 6 gene construct);    -   17. pDElia11-ask-asd-dapB-ORF2-dapA-ddh-lysA (pD11-KDABHL): ˜9.4        Kb PmeI fragment of pFC3-ask-asd-dapB-ORF2-dapA-ddh-lysA was        cloned into pDElia11 at the HincII site (contains the CAT gene;        is a full length 6 gene construct);    -   18. pDElia2: ˜1.24 Kb blunted PstI fragment of pUC4K ligated        with the ˜1.75 Kb DraI-SspI fragment of pUC 19;    -   19. pDElia11: ˜1 Kb PCR product containing the chloramphenicol        acyl-transferase gene expressed by the C. glutamicum fda        promoter was obtained using primers UCdraI and UCsspI and pM4 as        template and was ligated with the ˜1.75 Kb DraI-SspI fragment of        pUC19;

The primers utilized for the cloning procedures included:

-   -   ask: 5′-GGGTACCTCGCGAAGTAGCACCTGTCAC-3′ (SEQ ID NO:22)    -   asd: 5′-GCGGATCCCCCATCGCCCCTCAAAGA-3′ (SEQ ID NO:23)    -   dapB: 5′-AACGGGCGGTGAAGGGCAACT-3′ (SEQ ID NO:24)    -   dapA: 5′-TGAAAGACAGGGGTATCCAGA-3′ (SEQ ID NO:25)    -   ddh1: 5′-CCATGGTACCAAGTGCGTGGCGAG-3′ (SEQ ID NO:26)    -   ddh2: 5′-CCATGGTACCACACTGTTTCCTTGC-3′ (SEQ ID NO:27) KpnI        sites:GGTACC (SEQ ID NO:30)    -   argS: 5′-CTGGTTCCGGCGAGTGGAGCCGACCATTCCGCGAGG-3′ (SEQ ID NO:28)    -   lysA: 5′-CTCGCTCCGGCGAGGTCGGAGGCAACTTCTGCGACG-3′ (SEQ ID NO:29)    -   a primer that anneals internally to lysA (about 500 bp upstream        to the end of lysA).    -   UCdraI: 5′-GGATCTTCACCTAGATCC-3′ (SEQ ID NO:31)    -   UcsspI: 5′-CCCTGATAAATGCTTC-3′ (SEQ ID NO:32)

“K”, “D”, “A”, “B,” “H,” “L” and “′L” have the same designations as setforth above.

Example 5 Three-Fold Amplification of L-lysine Amino Acid BiosynthesisPathway Genes

For exemplary purposes only, Applicants provide herein an examplewherein at least one L-lysine amino acid biosynthesis pathway gene isamplified by a factor of 3.

Plasmid pD11-KDABH′L (constructed in Example 4) was used in theconstruction of high yield derivative cell lines of the invention. Forcell transformation experiments with the isolated nucleic acid moleculesof the invention, the growth preparation of competent cells, anddetermining of relative growth may be done according to the procedureset forth above.

Plasmid pD11-KDABH′L DNA was introduced into NRRL-B30220 (comprisingpK184-KDABH′L), using the electroporation method above. Introduction ofthe pD11-KDABH′L plasmid DNA into NRRL-B30220 resulted in incorporationof one copy of pD11-KDABH′L into the host cell chromosome via a singlecrossover homologous recombination event. The host cell comprising twocopies of five genes (pD11-KDABH′L and pK184-KDABH′L) has been depositedas NRRL-B30222.

The amount of lysine produced by C. glutamicum ATCC 21799 host cellshaving 3 copies of 5 genes (one endogenous copy and one copy of each ofpD11-KDABH′L and pK184-KDABH′L) is shown below.

L-lysine Production Strains L-lysine titer (g/L) L-lysine yield (%) ATCC21799 26.6 45.0 NRRL-B30222 32.0 56.0

Example 6

This example describes changing the promoter to increase the level ofexpression of each of these 6 genes described above. Six genes encodingsix different enyzmes of the biosynthetic pathway from L-aspartate toL-lysine have been inserted onto the chromosome of Corynebacteriumglutamicum. The additional copy of each gene is from a C. glutamicumstrain. The nucleotide sequences that regulate the level of expression(promoter) for each gene were the same as found on the C. glutamicumchromosome at the native loci.

Increased expression can result in increased specific activities of theenzymes and improved flux of carbon from aspartate to lysine. The yieldof lysine from glucose can be improved by this technique.

The level of expression from a promoter sequence is referred to asstrength. A strong promoter gives higher expression than a weak one. Themechanisms that determine the strength of a promoter have been described(Record, M. T., et al., “Escherichia coli RNA Polymerase, Promoters, andthe Kinetics of the Steps of Transcription Initiation,” in Escherichiacoli and Salmonella: Cellular and Molecular Biology, ASM Press (1996),pp. 792-881). Sources of promoters include nucleotide sequences from the5′ end of genes native to the C. glutamicum chromosome, from sequenceson plasmids that replicate in C. glutamicum, from sequences in thegenome of phage that infect C. glutamicum, or from sequences assembledby humans (tac, trc) and are not found in nature. Genes of ribosomalproteins, ribosomal RNAs and elongation factors show high levels ofexpression. The promoters of these genes are candidates for increasingexpression of amino acid biosynthetic pathway genes.

Another reason for changing promoters of genes in biosynthetic pathwaysis to make the pathway independent of factors that control the pathwayin the wild type organism. For example the native promoter of the operonthat contains diaminopimelate decarboxylase of the lysine biosyntheticpathway of C. glutamicum can respond to arginine or lysine in the growthmedium. Arginine increased transcription three-fold and lysine decreasedtranscription by one third (Oguiza, et al., J Bact.175:7356-7362(1993)). Diaminopimelate decarboxylase activity decreased60% in cells grown in minimal medium supplemented with 10 mmM lysine(Cremer et al., J Gen Microbiol. 134:3221-3229 (1988)). Replacing thepromoter of lysA which encodes the diaminopimelate decarboxylase is oneway to make lysine biosynthesis independent of arginine and lysinelevels in media.

Example 6A

Shown below are examples of promoters that are stronger than the askP1promoter which regulates the gene for aspartate kinase, the first enzymein the pathway from aspartate to lysine.

Beta-Galactosidase Assay of Candidate Promoters

Specific Activity Candidate micromol/min/mg Origin E12 0.20 no promoterE12/pTAC 49.80  pKK223-3 BF100 0.08 no promoter BF100/pAD151.1 2.22aspartokinase P1 E12 0.11 no promoter E12/pAD151.1 1.96 aspartokinase P1E12/5 3.46 BF100 genome E12/7 8.60 BF100 genome E12/10 6.56 BF100 genomeE12/32 3.11 BF100 genome E12/3 22.00  corynephage E12/39 11.57 corynephage E12/42 10.90  corynephageE12 is a C. glutamicum strain that does not produce lysine. E12 is alaboratory strain derived from ATCC 13059. BF100 is a high level lysineproducer (NRRL-B11474). TAC is commercially available promoter that hasbeen used as an example of a strong promoter. Four promoters from the C.glutamicum chromosome and three from a phage have been identified thatare stronger than the native aspartokinase promoter.

Example 6B

Examples of strong promoters increasing specific enzyme activity ofaspartokinase when expressed in C. glutamicum are shown below.

Influence of IPTG on Aspartokinase Activity

Regulator/ Strain promoter-gene Inducer nmol/min/mg BF100 none none 110PD9trc-ask lacI/trc-ask none 103 PD9trc-ask lacI/trc-ask +IPTG (30 mg/L)269 131-2 lacI/trc-ask none  59 131-2 lacI/trc-ask +IPTG (30 mg/L) 117131-5 lacI/trc-ask none  59 131-5 lacI/trc-ask +IPTG (30 mg/L) 123 pD9is a plasmid that replicates in C. glutamicum. 131 strains have thetrc-ask construct integrated into the genome. IPTG induces genescontrolled by the TRC promoter.

Example 6C

Examples of the influence of lacI/trc-ask on lysine production in shakeflasks are shown below.

Strain Induction O.D. Titre Yield S.P. BF100 none 46 26 43 58 PD9trc-asknone 49 30 49 61 PD9trc-ask +IPTG 45 30 50 68 BF100 none 43 23 39 53131-2 none 34 27 46 82 131-5 none 35 28 47 82 O.D. = optical density at660 nm Titre = grams Lysine/liter Yield = grams lysine made/gramsdextrose consumed S.P. = grams lysine/O.D.

The production of lysine by BF100 was improved by increasing thestrength of the aspartokinase promoter.

Example 7

This example demonstrates the use of vectorpDElia2-ask-asd-dapA-ORF2-dapB-ddh-P1lysA (pDElia2KDABHP1L) in theconstruction of the high yield cell lines of the invention. TheHpaI-PvaII fragment containing the P1 promoter was prepared as describedin Marcel T., et al., Molecular Microbiology 4:1819-1830 (1990).Applicants utilized standard PCR and subcloning procedures as set forthabove. For cell transformation experiments with the isolated nucleicacid molecules of the invention, the growth preparation of competentcells, and determining or relative growth may be done according to theprocedure set forth above.

Applicants performed the following steps in constructing the followingvectors used in the L-lysine biosynthetic pathway.

-   -   1. pGEMT-ask-asd: ˜2.6 Kb PCR product containing the ask-asd        operon of ATCC21529 using primers ask and asd was cloned into        pGEM-T (Promega pGEM-T vector systems).    -   2. pUC18-ddh: ˜1.3 KpnI fragment of pADM21 containing ddh (BF100        locus) was subcloned into pUC18 at the KpnI site.    -   3. pFC3-ask-asd: ˜2.6 Kb NsiI-ApaI fragment of pGEMT-ask-asd was        cloned into pFC3 cut with PstI and ApaI.    -   4. pFC3-dapB-ORF2-dapA: ˜2.9 Kb PCR product of NRRL-B11474        dapB-ORF2-dapA coding region was cloned into pFC3 at the EcoRV        site.    -   5. pFC1-ddh: ˜1.3 Kb PstI-EcoRI fragment of pUC18-ddh was cloned        into pFC1 cut with PstI and EcoRI.    -   6. pUC19-P1: ˜550 bp HpaI-PvuII fragment (containing the first        promoter, P1, of the argS-lysA operon) of pRS6 was cloned into        pUC19 at the SmaI site.    -   7. pUC19-P1lysA: ˜1.45 Kb promoterless PCR product, using primer        LysA(ATG) and LysA3B, of NRRL-B11474 lysA coding region is        cloned into pUC19-P1 at the HincII site.    -   8. pFC1-P1lysA: ˜2 Kb EcoRI-HindIII fragment of pUC19-P1lysA was        cloned into pFC1 cut with EcoRI and HindIII.    -   9. pFC1-P1lysA-ddh: ˜1.3 Kb EcoRI-NotI fragment of pFC1-ddh was        cloned into pFC1-P1lysA cut with EcoRI and NotI.    -   10. pFC1-ask-asd-ddh-P1lysA: ˜2.6 Kb SwaI-FseI fragment of        pFC3-ask-asd was cloned into pFC1-ddh-P1lysA cut with SwaI and        FseI.    -   11. pFC3-ask-asd-dapB-ORF2-dapA-ddh-P1lysA (pFC3-KDABHP1L): ˜5.9        Kb SpeI fragment of pFC1-ask-asd-ddh-P1lysA was cloned into        pFC3-dapB-ORF2-dapA at the SpeI site.    -   12. pDElia2-ask-asd-dapB-ORF2-dapA-ddh-P1lysA        (pDElia2-KDABHP1L): ˜8.8 Kb PmeI fragment of        pFC3-ask-asd-dapB-ORF2-dapA-ddh-P1lysA was cloned into pDElia2        at the HincII site.        Primers Used in PCR:    -   lysA (ATG): CCGGAGAAGATGTAACAATGGCTAC (SEQ ID NO:33)    -   LysA3B: CCTCGACTGCAGACCCCTAGACACC (SEQ ID NO:34)

The nucleotide sequence (SEQ ID NO:17) of the HpaI-PvuII fragmentcontaining the promoter P1 is shown in FIG. 20. Results of lysineproduction in NRRL-B11474 comprising thepDElia2-ask-asd-dapA-ORF2-dapB-ddh-P1lysA (pDElia2 KDABHP1L) constructare shown below.

lysine lysine yield Strain tested titer (%) cell deposit NRRL-B11474 3035   NRRL-B11474::pDElia2-KDABHP1L 37 42.5 NRRL B30359

Example 8

This example demonstrates the use of vectorpDElia2_(FC5)-ask-asd-dapB-ddh-lysA (pDElia2_(FC5)KDBHL) in theconstruction of the high yield cell lines of the invention. ThepDElia2_(FC5)KDBHL vector comprises a truncated ORF2 gene and lacks adapA gene. The ORF2 gene was cleaved at an internal ClaI site, removingthe 3′ region and the dapA gene. A promoterless lysA gene was obtainedfrom NRRL-B11474. For cell transformation experiments with the isolatednucleic acid molecules of the invention, the growth preparation ofcompetent cells, and determining of relative growth may be doneaccording to the procedure set forth above. Applicants performed thefollowing steps in constructing the following vectors used in theL-lysine biosynthetic pathway.

-   -   1. pGEMT-ask-asd: ˜2.6 Kb PCR product containing the ask-asd        operon of ATCC21529 using primers ask and asd was cloned into        pGEM-T (Promega pGEM-T vector systems).    -   2. pFC3-ask-asd: ˜2.6 Kb NsiI-ApaI fragment of pGEMT-ask-asd was        cloned into pFC3 cut with PstI and ApaI.    -   3. pFC3-dapB-ORF2-dapA: ˜2.9 Kb PCR product of NRRL-B11474        dapB-ORF2-dapA coding region was cloned into pFC3 at the EcoRV        site.    -   4. pFC3-dapB: the large ClaI fragment of pFC3-dapB-ORF2-dapA was        religated.    -   5. pUC18-ddh: ˜1.3 Kb KpnI fragment of pADM21 containing ddh        (NRRL-B11474 locus) was subcloned into pUC18 at the KpnI site.    -   6. pFC1-ddh: ˜1.3 Kb SalI-EcoRI fragment of pUC18-ddh was cloned        into pFC1 cut with SalI and EcoRI.    -   7. pFC1-ddh-lysA: ˜2.1 Kb EcoRI-PstI fragment (containing the        intact lysA DNA) of pRS6 was clone into pFC1-ddh cut with EcoRI        and PstI.    -   8. pFC1-ask-asd-ddh-lysA: ˜2.6 Kb SwaI-FseI fragment of        pFC3-ask-asd was cloned into pFC1-ddh-lysA cut with SwaI and        FseI.    -   9. pFC3-ask-asd-dapB-ddh-lysA: ˜6 Kb SpeI fragment of        pFC1-ask-asd-ddh-lysA was cloned into pFC3-dapB at the SpeI        site.    -   10. pDElia2_(FC5)-ask-asd-dapB-ddh-lysA (pDElia2_(FC5)-KDBHL):        ˜7.3 Kb NotI-PmeI fragment of pFC3-ask-asd-dapB-ddh-lysA was        cloned into pDElia2_(FC5) cut with NotI and PmeI.    -   11. pDElia2_(FC5): the small PvuII fragment of pFC5 was ligated        with the large PvuII fragment of pDElia2.

Results of lysine production in NRRL-B11474 comprising thepDElia2_(FC5)-ask-asd-dapB-ddh-lysA (pDElia2_(FC5)KDBHL) are shownbelow.

lysine lysine yield Strain tested titer (%) cell deposit NRRL-B1147431   49 NRRL-B11474::pDElia2_(FC5)-KDBHL 37.8 58 NRRL B30360

Having now fully described the present invention in some detail by wayof illustration and example for purposes of clarity of understanding, itwill be obvious to one of ordinary skill in the art that same can beperformed by modifying or changing the invention with a wide andequivalent range of conditions, formulations and other parametersthereof, and that such modifications or changes are intended to beencompassed within the scope of the appended claims.

All publications, patents and patent applications mentioned in thisspecification are indicative of the level of skill of those skilled inthe art to which this invention pertains, and are herein incorporated byreference to the same extent as if each individual publication, patentor patent application was specifically and individually indicated to beincorporated by reference.

1. An isolated polynucleotide molecule comprising a nucleotide sequenceencoding the polypeptide sequence of SEQ ID NO:2.
 2. The isolatedpolynucleotide molecule of claim 1 comprising a nucleic acid having thesequence of SEQ ID NO:1.
 3. A vector comprising the isolatedpolynucleotide molecule of claim
 1. 4. A host cell comprising the vectorof claim
 3. 5. The isolated polynucleotide molecule of claim 1 furthercomprising a promoter sequence where said promoter sequence has at least95% sequence identity to SEQ ID NO: 17, wherein said promoter sequencecontrols expression of said polynucleotide.
 6. The polynucleotide ofclaim 5 where said promoter sequence has the nucleotide sequence of SEQID NO:
 17. 7. A vector comprising the isolated polynucleotide of claim5.
 8. A host cell comprising the vector of claim
 7. 9. The host cell ofclaim 8 wherein said host cell is NRRL B
 30359. 10. A method fortransforming a Corynebacterium species host cell comprising: (a)transforming a Corynebacterium species host cell with the polynucleotidemolecule of claim 5, and (b) selecting a transformed host cell.
 11. Anisolated polynucleotide molecule comprising: (a) the polynucleotidemolecule of claim 1; and (b) at least one additional Corynebacteriumspecies lysine pathway gene selected from the group consisting of: (i) anucleic acid molecule encoding the asd polypeptide of SEQ ID NO:4; (ii)a nucleic acid molecule encoding the dapA polypeptide of SEQ ID NO:6;(iii) a nucleic acid molecule encoding the dapB polypeptide of SEQ IDNO:8; (iv) a nucleic acid molecule encoding the ddh polypeptide of SEQID NO:10; (v) a nucleic acid molecule encoding the ′lysA polypeptide ofSEQ ID NO:21; (vi) a nucleic acid molecule encoding the lysA polypeptideof SEQ ID NO:14; and (vii) a nucleic acid molecule encoding the ORF2polypeptide of SEQ ID NO:16.
 12. A vector comprising the polynucleotidemolecule of claim
 11. 13. A host cell comprising the vector of claim 12.14. The host cell of claim 13 wherein said host cell is a Brevibacteriumflavum selected from the group consisting of Brevibacterium flavumNRRL-B30218, Brevibacterium flavum NRRL-B30219, Brevibacteriumlactofermentum NRRL-B30220, Brevibacterium lactofermentum NRRL-B30221,Brevibacterium lactofermentum NRRL-B30222, Brevibacterium flavumNRRL-B30234 and Brevibacterium lactofermentum NRRL-B30235.
 15. The hostcell of claim 13 wherein said host cell is Escherichia coli DH5 α MCRNRRL-B30228.
 16. The host cell of claim 13 wherein said host cell is aC.glutamicum selected from the group consisting of C.glutamicumNRRL-B30236 and C.glutamicum NRRL-B30237.
 17. The isolatedpolynucleotide of claim 11, wherein said additional Corynebacteriumspecies lysine pathway gene encodes the asd polypeptide of SEQ ID NO:4.18. The isolated polynucleotide of claim 11, wherein said additionalCorynebacterium species lysine pathway gene encodes the dapA polypeptideof SEQ ID NO:6.
 19. The isolated polynucleotide of claim 11, whereinsaid additional Corynebacterium species lysine pathway gene encodes thedapB polypeptide of SEQ ID NO:8.
 20. The isolated polynucleotide ofclaim 11, wherein said additional Corynebacterium species lysine pathwaygene encodes the ddh polypeptide of SEQ ID NO:10.
 21. The isolatedpolynucleotide of claim 11, wherein said additional Corynebacteriumspecies lysine pathway gene encodes the ′lysA polypeptide of SEQ IDNO:21.
 22. The isolated polynucleotide of claim 11, wherein saidadditional Corynebacterium species lysine pathway gene encodes the lysApolypeptide of SEQ ID NO:14.
 23. The isolated polynucleotide of claim11, wherein said additional Corynebacterium species lysine pathway geneencodes the ORF2 polypeptide of SEQ ID NO:16.
 24. An isolatedpolynucleotide molecule comprising: (a) the polynucleotide molecule ofclaim 1; (b) a nucleic acid molecule encoding the asd amino acidsequence of SEQ ID NO:4; (c) a nucleic acid molecule encoding the dapAamino acid sequence of SEQ ID NO:6; (d) a nucleic acid molecule encodingthe dapB amino acid sequence of SEQ ID NO:8; and (e) a nucleic acidmolecule encoding the ORF2 amino acid sequence of SEQ ID NO:16.
 25. Theisolated polynucleotide molecule of claim 24 comprising pK 184-KDAB. 26.An isolated polynucleotide molecule comprising: (a) the polynucleotidemolecule of claim 1; (b) a nucleic acid molecule encoding the asd aminoacid sequence of SEQ ID NO:4; (c) a nucleic acid molecule encoding thedapA amino acid sequence of SEQ ID NO:6; (d) a nucleic acid moleculeencoding the dapB amino acid sequence of SEQ ID NO:8; (e) a nucleic acidmolecule encoding the ddh amino acid sequence of SEQ ID NO:10; and (f) anucleic acid molecule encoding the ORF2 amino acid sequence of SEQ IDNO:16.
 27. An isolated polynucleotide molecule comprising: (a) thepolynucleotide molecule of claim 1; (b) a nucleic acid molecule encodingthe asd amino acid sequence of SEQ ID NO:4; (c) a nucleic acid moleculeencoding the dapA amino acid sequence of SEQ ID NO:6; (d) a nucleic acidmolecule encoding the dapB amino acid sequence of SEQ ID NO:8; (e) anucleic acid molecule encoding the ddh amino acid sequence of SEQ IDNO:10; (f) a nucleic acid molecule encoding the ′lysA amino acidsequence of SEQ ID NO:21; and (g) a nucleic acid molecule encoding theORF2 amino acid sequence of SEQ ID NO:16.
 28. The isolatedpolynucleotide molecule of claim 27 comprising pD11-KDABH′L.
 29. Anisolated polynucleotide molecule comprising: (a) the polynucleotidemolecule of claim 2; (b) a nucleic acid molecule encoding the asd aminoacid sequence of SEQ ID NO:4; (c) a nucleic acid molecule encoding thedapA amino acid sequence of SEQ ID NO:6; (d) a nucleic acid moleculeencoding the dapB amino acid sequence of SEQ ID NO:8; (e) a nucleic acidmolecule encoding the ddh amino acid sequence of SEQ ID NO:10; (f) anucleic acid molecule encoding the lysA amino acid sequence of SEQ IDNO:14; and (g) a nucleic acid molecule encoding the ORF2 amino acidsequence of SEQ ID NO:16.
 30. The isolated polynucleotide molecule ofclaim 29 comprising pD2-KDABHL.
 31. A method for transforming aCorynebacterium species host cell comprising: (a) transforming aCorynebacterium species host cell with an isolated polynucleotidemolecule comprising a nucleotide sequence encoding the polypeptide ofSEQ ID NO:2 and (b) selecting a transformed host cell.
 32. The method ofclaim 31 further comprising screening for said transformedpolynucleotide molecule.
 33. The method of claim 31 further comprising:(a) growing said transformed host cell in a medium; and (b) purifying anamino acid produced by said transformed host cell.
 34. The method ofclaim 31 wherein the nucleotide sequence is integrated into said hostcell's chromosome.
 35. The method of claim 31 wherein said host cellpossesses at least one of the following activities: (a)aspartate-semialdehyde dehydrogenase activity; (b) dihydrodipicolinatesynthase activity; (c) dihydrodipicolinate reductase activity; (d)diaminopimelate dehydrogenase activity; and (e) diaminopimelatedecarboxylase activity.
 36. The method of claim 35 further comprisingscreening for said activity.
 37. The method of claim 35 wherein saidactivity is aspartate-semialdehyde dehydrogenase activity.
 38. Themethod of claim 37, wherein said aspartate-semialdehyde dehydrogenaseactivity is produced by the asd polypeptide encoded by thepolynucleotide of SEQ ID NO:3.
 39. The method of claim 35 wherein saidactivity is dihydrodipicolinate synthase activity.
 40. The method ofclaim 39, wherein said dihydrodipicolinate synthase activity is producedby the dapA polypeptide encoded by the polynucleotide of SEQ ID NO:5.41. The method of claim 35 wherein said activity is dihydrodipicolinatereductase activity.
 42. The method of claim 41, wherein saiddihydrodipicolinate reductase activity is produced by the dapBpolypeptide encoded by the polynucleotide of SEQ ID NO:7.
 43. The methodof claim 35 wherein said activity is diaminopimelate dehydrogenaseactivity.
 44. The method of claim 43, wherein said diaminopimelatedehydrogenase activity is produced by the ddh polypeptide encoded by thepolynucleotide of SEQ ID NO:9.
 45. The method of claim 35 wherein saidactivity is diaminopimelate decarboxylase activity.
 46. The method ofclaim 45, wherein said diaminopimelate decarboxylase activity isproduced by the ′lysA polypeptide encoded by the polynucleotide of SEQID NO:20.
 47. The method of claim 45, wherein said diaminopimelatedecarboxylase activity is produced by the lysA polypeptide encoded bythe polynucleotide of SEQ ID NO:13.
 48. The method of claim 31, whereinsaid isolated polynucleotide molecules further comprises at least onenucleic acid molecule selected from the group consisting of: a) anucleic acid molecule encoding the asd amino acid sequence of SEQ IDNO:4; b) a nucleic acid molecule encoding the dapA amino acid sequenceof SEQ ID NO:6; c) a nucleic acid molecule encoding the dapB amino acidsequence of SEQ ID NO:8; d) a nucleic acid molecule encoding the ddhamino acid sequence of SEQ ID NO:10; e) a nucleic acid molecule encodingthe ′lysA amino acid sequence of SEQ ID NO:21; f) a nucleic acidmolecule encoding the lysA amino acid sequence of SEQ ID NO:14; and (g)a nucleic acid molecule encoding the ORF2 amino acid sequence of SEQ IDNO:16.
 49. The method of claim 31, wherein said isolated polynucleotidemolecule further comprises the following: (a) a nucleic acid moleculeencoding the asd amino acid sequence of SEQ ID NO:4; (b) a nucleic acidmolecule encoding the dapA amino acid sequence of SEQ ID NO:6; (c) anucleic acid molecule encoding the dapB amino acid sequence of SEQ IDNO:8; and (d) a nucleic acid molecule encoding the ORF2 amino acidsequence of SEQ ID NO:16.
 50. The method of claim 31, wherein saidisolated polynucleotide molecule further comprises the following: (a) anucleic acid molecule encoding the asd amino acid sequence of SEQ IDNO:4; (b) a nucleic acid molecule encoding the dapA amino acid sequenceof SEQ ID NO:6; (c) a nucleic acid molecule encoding the dapB amino acidsequence of SEQ ID NO:8; (d) a nucleic acid molecule encoding the ddhamino acid sequence of SEQ ID NO: 10; and (e) a nucleic acid moleculeencoding the ORF2 amino acid sequence of SEQ ID NO:16.
 51. The method ofclaim 31, wherein said isolated polynucleotide molecule furthercomprises the following: (a) a nucleic acid molecule encoding the asdamino acid sequence of SEQ ID NO:4; (b) a nucleic acid molecule encodingthe dapA amino acid sequence of SEQ ID NO:6; (c) a nucleic acid moleculeencoding the dapB amino acid sequence of SEQ ID NO:8; (d) a nucleic acidmolecule encoding the ddh amino acid sequence of SEQ ID NO:10; (e) anucleic acid molecule encoding the ′lysA amino acid sequence of SEQ IDNO:21; and (f) a nucleic acid molecule encoding the ORF2 amino acidsequence of SEQ ID NO:16.
 52. The method of claim 31, wherein saidisolated polynucleotide molecule further comprises the following: (a) anucleic acid molecule encoding the asd amino acid sequence of SEQ IDNO:4; (b) a nucleic acid molecule encoding the dapA amino acid sequenceof SEQ ID NO:6; (c) a nucleic acid molecule encoding the dapB amino acidsequence of SEQ ID NO:8; (d) a nucleic acid molecule encoding the ddhamino acid sequence of SEQ ID NO:10; (e) a nucleic acid moleculeencoding the lysA amino acid sequence of SEQ ID NO:14; and (f) a nucleicacid molecule encoding the ORF2 amino acid sequence of SEQ ID NO:16.